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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    Der Ophthalmologe 97 (2000), S. 758-763 
    ISSN: 1433-0423
    Keywords: Schlüsselwörter LASIK ; PRK ; Deutschland ; Operationszahlen ; Krankenkassen ; Keywords LASIK ; PRK ; Dissemination ; German sickness funds
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Abstract Background. Little information about the cost-effectiveness of excimer laser operations is available. As the number and structure of providers of these services in Germany are relatively unknown, only rough estimations can be made about the number of operations. Purpose. In this study the market for excimer laser operations is defined, structured from an economic view and examined according to medium-term demand and supply trends. The aim of the study is an applicable estimation of the current level of dissemination and of existing economic conditions for providers of excimer laser operations. Methods. In a postal survey 219 ophthalmologists in Germany were asked to provide the number of excimer laser operations they had carried out and the organizational and financial details of these services. The questionnaire was answered anonymously. Results. One can conclude that the annual number of interventions is increasing, although less significantly than in the United States. In most cases, providers of German health insurance have rejected inclusion of this service in their reimbursement catalogue. Conclusions. As of yet, excimer-laser-related turnover has frequently not met expectations. However, because of modifications of technological, health-economic and demand conditions an increase in the number of operations within this area is expected in the future.
    Notes: Zusammenfassung Hintergrund. Zur Kosteneffektivität von Excimer-Laser-Operationen liegen bislang kaum Informationen vor. Auch die Anzahl und Struktur der Anbieter dieser Leistungen in Deutschland sind weitgehendst unbekannt. Es liegen nur grobe Schätzungen zur Anzahl der Operationen vor. In dieser Studie wird der Markt für Excimer-Laser-Operationen aus ökonomischer Sicht abgegrenzt, strukturiert und bezüglich mittelfristiger Nachfrage- und Anbietertrends untersucht. Ziel der Arbeit ist eine zutreffende Abschätzung der derzeitigen Verbreitung und der ökonomischen Rahmenbedingungen bei Anbietern von Excimer-Laser-Operationen. Methode. 219 Augenärzte im gesamten Bundesgebiet wurden postalisch danach befragt, in welchem Umfang sie Excimer-Laser-Operationen durchführen und welche organisatorischen und finanziellen Rahmenbedingungen dabei bestehen. Die Beantwortung erfolgte anonym. Ergebnisse. Insgesamt ist ein Ansteigen der jährlichen Anzahl der Eingriffe festzustellen, jedoch weniger ausgeprägt als in den USA. Eine Aufnahme dieser Leistung in den Erstattungskatalog der Krankenkassen wird von den Anbietern überwiegend abgelehnt. Schlussfolgerungen. Die mit einem Excimer-Laser verbundenen Umsatzerwartungen sind bislang häufig nicht erfüllt worden. Änderungen technologischer und gesundheitspolitischer Rahmenbedingungen sowie die steigende Nachfrage deuten aber darauf hin, dass zukünftig die Operationszahlen in diesem Bereich nachhaltig ansteigen werden.
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    Der Nervenarzt 71 (2000), S. 552-558 
    ISSN: 1433-0407
    Keywords: Schlüsselwörter Epidemiologie ; Bulimia nervosa ; Anorexia nervosa ; “Binge-eating-Störung” ; Studierende ; Fragebogen ; Deutschland ; Key words Epidemiology ; Bulimia nervosa ; Anorexia nervosa ; Binge-eating disorder ; Students ; Questionnaire ; Germany
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Abstract A sample of 507 social work students completed the Bulimic Investigatory Test Edinburgh (BITE). Simulating diagnoses according to DSM-IV criteria, we found three women suffering from bulimia nervosa (BN). This represents a total prevalence of 0.6%, 0.8% in women, and 0.9% in female probands up to the age of 30 years. In the same way, we identified one case of anorexia nervosa (AN), i. e. a total prevalence of 0.2%, 0,3% in women, and 0.3% in female probands up to the age of 30. Nineteen students also fulfilled DSM-IV research criteria for binge-eating disorder (BED), showing a total prevalence of 3.7%, 3.8% in women, 3.5% in men, and 4.3% in female probands up to the age of 30. Thus, BED is the most common eating disorder and also occurs in men. In light of the association between weight discontent and eating disorders, suggestions are made for the management of overweight patients and both normal and underweight clients with eating disorders.
    Notes: Zusammenfassung Von 507 Studierenden der Sozialarbeit oder -pädagogik wurde der Bulimic Investigatory Test Edinburgh (BITE) ausgefüllt. Anhand von nach DSM-IV-Kriterien simulierten Diagnosen fanden wir 3 Frauen, die vermutlich an einer Bulimia nervosa (BN) litten. Das bedeutet eine Punktprävalenz für die Gesamtstichprobe von 0,6%, für alle Frauen von 0,8% und von 0,9% für Frauen bis zu 30 Jahren. Weiterhin identifizierten wir 19 Personen (3,7%), die DSM-IV-Forschungskriterien für die “Binge-eating-Störung” (“binge-eating disorder”, BED) erfüllten, 4 von 113 Männern (3,5%), 15 von 394 Frauen (3,8%) und 14 von 329 Frauen bis zu 30 Jahren (4,3%). DSM-IV-Anorexia nervosa (AN) entdeckten wir auf dieselbe Weise bei einer 30-jährigen Studentin. Das bedeutet eine Punktprävalenz für die Gesamtstichprobe von 0,2%, für alle Frauen von 0,3% und von 0,3% für Frauen bis zu 30 Jahren. Damit ist BED die häufigste Essstörung, und sie kommt auch bei Männern vor. Wegen des Zusammenhangs zwischen Unzufriedenheit mit dem Gewicht und gestörtem Essverhalten werden Hinweise für den Umgang mit Übergewichtigen generell sowie normal- und untergewichtigen Essgestörten gegeben.
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    ISSN: 1437-1588
    Keywords: Schlüsselwörter Tuberkulose ; Epidemiologie ; Deutschland ; Osteuropa ; Weltweit ; Resistenzsituation ; DOTS ; Key words Tuberculosis ; Epidemiology ; Germany ; Eastern Europe ; Worldwide ; Resistance situation ; DOTS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary From a global viewpoint, tuberculosis is one of the most important infectious diseases of our time. The situation in Germany, as in most comparable industrialized nations, is stable. The declining trend of previous years continued in 1997, when 11,163 people developed active tuberculosis – an incidence of 13.6/100,000. However, the tuberculosis problem cannot be restricted to one country, and Germany is particularly affected by trends in Eastern Europe and in the former Soviet countries. These countries experienced a marked increase of tuberculosis cases and resistant strains during recent years, a tendency which can only be countered by fast, well-aimed and efficient action in the affected areas. The industrialized nations in particular should consider financial and logistic contributions as their duty. Crucial for control of the tuberculosis situation within each country are registration and close observation of epidemiological trends, identification of high-risk groups, and continuation of established tuberculosis control measures.
    Notes: Zusammenfassung Global gesehen ist die Tuberkulose heutzutage eine der wichtigsten Infektionskrankheiten. In Deutschland, wie auch in den meisten vergleichbaren Industrienationen, ist die Situation stabil. 1997 setzte sich der rückläufige Trend der letzten Jahre fort; es erkrankten 11.163 Menschen an einer aktiven Tuberkulose, entsprechend einer Inzidenz von 13,6/100.000. Die Tuberkulose ist jedoch ein grenzüberschreitendes Problem, insbesondere die Entwicklung in Osteuropa und den Ländern der ehemaligen Sowjetunion ist für Deutschland von Bedeutung. Hier zeigt sich in den letzten Jahren ein deutlicher Anstieg der Tuberkulosefälle und zudem auch eine Zunahme resistenter Erreger. Nur durch rasches, gezieltes und effizientes Handeln vor Ort besteht die Möglichkeit, diesem Trend entgegenzuwirken. Insbesondere die Industrienationen sind gefordert, finanziell und logistisch Unterstützung zu leisten. Entscheidend für die Kontrolle der Tuberkulosesituation im eigenen Land ist die Erfassung und aufmerksame Beobachtung aktueller epidemiologischer Trends, die Identifikation gefährdeter Personengruppen sowie die Aufrechterhaltung bewährter Tuberkulose-Bekämpfungsmaßnahmen.
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  • 4
    Electronic Resource
    Electronic Resource
    Springer
    Der Hautarzt 49 (1998), S. 826-834 
    ISSN: 1432-1173
    Keywords: Schlüsselwörter Malignes Melanom ; Prävention ; Australien ; USA ; Großbritannien ; Deutschland ; Key words Cutaneous Malignant Melanoma ; Prevention ; Australia ; USA ; Great Britain ; Germany
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine
    Description / Table of Contents: Summary Dermatologists and physicians of other specialities, as well as other health professionals have made tremendous efforts to improve the public education (primary prevention) and early detection (secondary prevention) of cutaneous malignant melanoma (CMM) especially during the last decade. In Australia, the country with the highest incidence of CMM in the world, the first public and effective campaigns were already carried out in the sixties. Through the public campaigns, the knowledge increased about skin cancer, and the attitude and behavior toward sun exposure changed in the population. In the USA and Great Britain too, effective public campaigns were carried out in great numbers and extensiveexperience was acquired. In Germany, prevention campaigns were first run in regional areas. In 1989, the Commission of Early Detection and Prevention of Melanoma of the German Dermatological Society launched nation-wide campaigns. These activities were complemented by regional campaigns in the 1990s. The analysis of previous campaigns demonstrates that single activities are less effective and repeated campaigns are necessary to increase knowledge about skin and to change attitudes and behavior towards UV-exposure. In addition, the development of sun protective clothings and structural changes, e.g. creation of shady places around open-air swimming pools, should be aimed for. Physicians of other specialities and other health professionals should also be included in prevention campaigns.
    Notes: Zusammenfassung Beim malignen Melanom der Haut wurden insbesondere im letzten Jahrzehnt von Dermatologen und Ärzten anderer Fachrichtungen sowie weiteren Angehörigen von Gesundheitsberufen große Anstrengungen unternommen, die Aufklärung (primäre Prävention) und Früherkennung (sekundäre Prävention) zu verbessern. Die erste öffentlichkeitswirksame Kampagne zur Prävention des malignen Melanoms wurde bereits in den 60er Jahren in Australien, dem Land mit der weltweit höchsten Melanominzidenz, durchgeführt. Durch Aufklärung vermehrte sich das Wissen über Hautkrebs, und Einstellung und Verhalten der Bevölkerung in bezug auf die Sonne änderten sich. Auch in den USA und in Großbritannien wurden zahlreiche öffentlichkeitswirksame Aktivitäten durchgeführt und umfangreiche Erfahrungen gesammelt. In Deutschland wurden Aufklärungsaktionen zunächst auf regionaler Ebene durchgeführt. 1989 initiierte die Kommission zur Früherkennung und Prävention des Melanoms der Deutschen Dermatologischen Gesellschaft bundesweite Kampagnen. Regionale Aktionen ergänzten diese Aktivitäten in den 90er Jahren. Die Analyse bisheriger Aktionen zeigt, daß einmalige Aktivitäten wenig wirksam und daß Wiederholungen von öffentlichkeitswirksamen Präventionskampagnen notwendig sind, um das Wissen in der Bevölkerung über Hautkrebs zu verbessern und Einstellungen und Verhalten gegenüber der UV-Exposition zu ändern. Ergänzend hierzu sollten die Entwicklung von Textilien mit hohem Lichtschutzfaktor und strukturelle Veränderungen, z.B. Schaffung von Schattenplätzen in Schwimmbädern, angestrebt werden. In Präventionskampagnen sollten auch vermehrt Ärzte anderer Disziplinen sowie weitere Gesundheitsberufe mit einbezogen werden.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: residue location parameter ; environment parameter ; protein fold description ; protein fold recognition ; threading ; homogeneity ; amino acid type discrimination ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The parametric description of residue environments through solvent accessibility, backbone conformation, or pairwise residue-residue distances is the key to the comparison between amino acid types at protein sequence positions and residue locations in structural templates (condition of protein sequence-structure match). For the first time, the research results presented in this study clarify and allow to quantify, on a rigorous statistical basis, to what extent the amino acid type-specific distributions of commonly used environment parameters are discriminative with respect to the 20 amino acid types. Relying on the Bahadur theory, we estimate the probability of error in a single-sequence-structure alignment based on weak or absent discriminative power in a learning database of protein structure. We present the results for many residue environment variables and demonstrate that each fold description parameter is sensitive with respect to only a few amino acid types while indifferent to most of the other amino acid types. Even complex structural characteristics combining solvent-accessible surface area, backbone conformation, and pairwise distances distinguish only some amino acid types, whereas the others remain nondiscriminated. We find that the knowledge-based potentials currently in use treat especially Ala, Asp, Gln, His, Ser, Thr, and Tyr as essentially “average” amino acids. Thus, highly discriminative amino acid types define the alignment register in gapless sequence-structure alignments. The introduction of gaps leads to alignment ambiguities at sequence positions occupied by nondiscriminated amino acid types. Therefore, local sequence-structure alignments produced by techniques with gaps cannot be reliable. Conceptionally new and more sensitive environment parameters must be invented. Proteins 31:225-246, 1998. © 1998 Wiley-Liss, Inc.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: homology modeling ; database searching ; conserved torsional angles ; prediction of sidechain conformations ; homologous families of proteins ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We investigated the conservation of sidechain conformation for each residue within a homologous family of proteins in the Protein Data Bank (PDB) and performed sidechain modeling using this information. The information was represented by the probability of conserved sidechain torsional angles obtained from many families of proteins, and these were calculated for a pair of residues at topologically equivalent positions as a result of structural alignment. Probabilities were obtained for a pair of same amino acids and for a pair of different amino acids. The correlation between environmental residues and the fluctuation of probability was examined for the pair of same amino acid residues, and the simple probability was calculated for the pair of different amino acids. From the results on the same amino acid pairs, 17 amino acids, except for Ala, Gly, and Pro, were divided into two types: those that were influenced and those that were not influenced by the environmental residues. From results on different amino acid pairs, a replacement between large residues, such as Trp, Phe, and Tyr, was performed assuming conservation of their torsional angles within a homologous family of proteins. We performed sidechain modeling for 11 known proteins from their native and modeled backbones, respectively. With the native backbones, the percentage of the χ1 angle correct within 30° was found to be 67% and 80% for all and core residues, respectively. With the modeled backbones, the percentage of the correct χ1 angle was found to be 60% and 72% for all and core residues, respectively. To estimate an upper limit on the accuracy for predicting sidechain conformations, we investigated the probability of conserved sidechain torsional angles for highly similar proteins having 〉 90% sequence identity and 〈2.5-Å X-ray resolution. In those proteins, 83% of the sidechain conformations were conserved for the χ1 angle. Proteins 31:355-369, 1998. © 1998 Wiley-Liss, Inc.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: NMR structure refinement ; correlated/collective motion ; essential dynamics analysis ; PH domain ; single-stranded DNA binding protein ; gene V protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Large concerted motions of proteins which span its “essential space,” are an important component of protein dynamics. We investigate to what extent structure ensembles generated with standard structure calculation techniques such as simulated annealing can capture these motions by comparing them to long-time molecular dynamics (MD) trajectories. The motions are analyzed by principal component analysis and compared using inner products of eigenvectors of the respective covariance matrices. Two very different systems are studied, the β-spectrin PH domain and the single-stranded DNA binding protein (ssDBP) from the filamentous phage Pf3. A comparison of the ensembles from NMR and MD shows significant overlap of the essential spaces, which in the case of ssDBP is extraordinarily high. The influence of variations in the specifications of distance restraints is investigated. We also study the influence of the selection criterion for the final structure ensemble on the definition of mobility. The results suggest a modified criterion that improves conformational sampling in terms of amplitudes of correlated motion. Proteins 31:370-382, 1998. © 1998 Wiley-Liss, Inc.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: time-resolved small-angle X-ray scattering ; allosterism ; domain closure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Time-resolved small-angle X-ray scattering (TR-SAXS) was used to monitor the structural changes that occur upon the binding of the natural substrates to a mutant version of the allosteric enzyme aspartate transcarbamoylase from Escherichia coli, in which the creation of a critical link stabilizing the R state of the enzyme is hindered. Previously, SAXS experiments at equilibrium showed that the structures of the unligated mutant enzyme and the mutant enzyme saturated with a bisubstrate analog are indistinguishable from the T and R state structures, respectively, of the wild-type enzyme (Tauc et al., Protein Sci. 3:1998-2004, 1994). However, as opposed to the wild-type enzyme, the combination of one substrate, carbamoyl phosphate, and succinate, an analog of aspartate, did not convert the mutant enzyme into the R state. By using TR-SAXS we have been able to study the transient steady-state during catalysis using the natural substrates rather than the nonreactive substrate analogs. The steady-state in the presence of saturating amount of substrates is a mixture of 60% T and 40% R structures, which is further converted entirely to R in the additional presence of ATP. These results provide a structural explanation for the reduced cooperativity observed with the mutant enzyme as well as for the stimulation by ATP at saturating concentrations of substrates. They also illustrate the crucial role played by domain motions and quaternary-structure changes for both the homotropic and heterotropic aspects of allostery. Proteins 31:383-390, 1998. © 1998 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: complement control protein ; protein modeling ; blood coagulation ; C4b-binding protein ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: C4b-binding protein (C4BP) contributes to the regulation of the classical pathway of the complement system and plays an important role in blood coagulation. The main human C4BP isoform is composed of one β-chain and seven α-chains essentially built from three and eight complement control protein (CCP) modules, respectively, followed by a nonrepeat carboxy-terminal region involved in polymerization of the chains. C4BP is known to interact with heparin, C4b, complement factor I, serum amyloid P component, streptococcal Arp and Sir proteins, and factor VIII/VIIIa via its α-chains and with protein S through its β-chain. The principal aim of the present study was to localize regions of C4BP involved in the interaction with C4b, Arp, and heparin. For this purpose, a computer model of the 8 CCP modules of C4BP α-chain was constructed, taking into account data from previous electron microscopy (EM) studies. This structure was investigated in the context of known and/or new experimental data. Analysis of the α-chain model, together with monoclonal antibody studies and heparin binding experiments, suggests that a patch of positively charged residues, at the interface between the first and second CCP modules, plays an important role in the interaction between C4BP and C4b/Arp/Sir/heparin. Putative binding sites, secondary-structure prediction for the central core, and an overall reevaluation of the size of the C4BP molecule are also presented. An understanding of these intermolecular interactions should contribute to the rational design of potential therapeutic agents aiming at interfering specifically some of these protein-protein interactions. Proteins 31:391-405, 1998. © 1998 Wiley-Liss, Inc.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: electrostatics ; Brownian dynamics ; triose phosphate isomerase ; diffusion-control ; similarity index ; rate enhancement ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Triose phosphate isomerase (TIM) is a diffusion-controlled enzyme whose rate is limited by the diffusional encounter of the negatively charged substrate glyceraldehyde 3-phosphate (GAP) with the homodimeric enzyme's active sites. Translational and orientational steering of GAP toward the active sites by the electrostatic field of chicken muscle TIM has been observed in previous Brownian dynamics (BD) simulations. Here we report simulations of the association of GAP with TIMs from four species with net charges at pH 7 varying from -12e to +12e. Computed second-order rate constants are in good agreement with experimental data. The BD simulations and computation of average Boltzmann factors of substrate-protein interaction energies show that the protein electrostatic potential enhances the rates for all the enzymes. There is much less variation in the computed rates than might be expected on the basis of the net charges. Comparison of the electrostatic potentials by means of similarity indices shows that this is due to conservation of the local electrostatic potentials around the active sites which are the primary determinants of electrostatic steering of the substrate. Proteins 31:406-416, 1998. © 1998 Wiley-Liss, Inc.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: molecular evolution ; protein evolution ; mutation matrices ; Metropolis kinetics ; Boltzmann statistics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: New computational models of natural site mutations are developed that account for the different selective pressures acting on different locations in the protein. The number of adjustable parameters is greatly reduced by basing the models on the underlying physical-chemical properties of the amino acids. This allows us to use our method on small data sets built of specific protein types. We demonstrate that with this approach we can represent the evolutionary patterns in HIV envelope proteins far better than with more traditional methods. Proteins 32:289-295, 1998. © 1998 Wiley-Liss, Inc.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: photon absorption simulation ; SCF-CI ; chromophore ; vertical transition ; conformational change ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecular dynamics simulations were carried out to study what happens in a photoreceptor protein, photoactive yellow protein (PYP), immediately after the vertical transition of the chromophore from the ground to the excited state. A photon absorption simulation was performed to investigate the movement of amino acid residues upon photoexcitation. To calculate the excited state of the chromophore, SCF-CI calculation was carried out with INDO/S Hamiltonian. We observed that some amino acid residues have strong interactions with the chromophore. Most of these amino acid residues are conserved in PYPs from three different species of bacteria. This observation indicates the biological importance of these residues. Proteins 32:268-275, 1998. © 1998 Wiley-Liss, Inc.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: adenylate kinase ; Mg2+ and Mn2+ coordination ; zinc fingers ; entropic substrate release ; thermostability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Crystal structures of Bacillus stearothermophilus adenylate kinase with bound Ap5A, Mn2+ Ap5A, and Mg2+ Ap5A have been determined by X-ray crystallography to resolutions of 1.6 Å, 1.85 Å, and 1.96 Å, respectively. The protein's lid domain is partially open, being both rotated and translated away from bound Ap5A. The flexibility of the lid domain in the ternary state and its ability to transfer force directly to the the active site is discussed in light of our proposed entropic mechanism for catalytic turnover. The bound Zn2+ atom is demonstrably structural in nature, with no contacts other than its ligating cysteine residues within 5 Å. The B. stearothermophilus adenylate kinase lid appears to be a truncated zinc finger domain, lacking the DNA binding finger, which we have termed a zinc knuckle domain. In the Mg2+ Ap5A and Mn2+ Ap5A structures, Mg2+ and Mn2+ demonstrate six coordinate octahedral geometry. The interactions of the Mg2+-coordinated water molecules with the protein and Ap5A phosphate chain demonstrate their involvement in catalyzing phosphate transfer. The protein selects for β-γ (preferred by Mg2+) rather than α-γ (preferred by Mn2+) metal ion coordination by forcing the ATP phosphate chain to have an extended conformation. Proteins 32:276-288, 1998. © 1998 Wiley-Liss, Inc.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: normal mode analysis of a complex ; subtilisin-eglin c complex ; dynamics of a complex ; binding free energy ; internal and external motions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Normal mode analysis of subtilisin-eglin c complex was performed to investigate the dynamics at the interface between the enzyme and the inhibitor. The internal motions of the complex calculated from the normal modes were divided into three parts: the internal motions changing the shape of each molecule, the external rigid-body motions changing their mutual dispositions, and the coupling between the internal and external motions. From the results of the analysis, the following characteristic features were found in the dynamics at the interface regions: 1) negative correlation between the internal and external motions within each molecule, and 2) positive correlation between the external motions of the two molecules. The former decreases the apparent amplitudes of motions at the interface. The latter minimizes the interference between individual motions of the two molecules. These dynamic characteristics allow the enzyme and the inhibitor to move as freely as possible. This finding suggests that the experimental evidence of the large entropy gain on binding should be attributed not only to strong hydrophobic interactions, but also to the dynamic structure of the complex, which is found to minimize an unavoidable loss of the conformational entropy on binding. Proteins 32:324-333, 1998. © 1998 Wiley-Liss, Inc.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: AMBER ; amphipathic helix ; distance geometry ; DYANA ; fibrillation ; NMR ; sodium dodecyl sulfate ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), 3JHNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure-activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314-323, 1998. © 1998 Wiley-Liss, Inc.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: acid denatured state ; ANS fluorescence ; Arrhenius plot ; kinetics ; molten globule intermediate ; TFE denatured state ; protein folding ; human stefin B ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: It has been shown that human stefin B exhibits molten globule intermediates when denatured by acid or GuHCl. In the presence of TFE, it transforms into a highly helical state. In our first study on its folding mechanism (Žerovnik et al., Proteins 32:296-303), the kinetics measured by circular dichroism (CD) and fluorescence were correlated. In the present work the kinetics of folding were monitored by tyrosine fluorescence, ANS fluorescence, and, for certain reactions, far ultraviolet (UV) CD. The folding was started from the unfolded state in 3.45 M GuHCl, the acid denatured state at pH 1.8 ± 0.2, an acid molten globule intermediate I1 (pH 3.3 ± 0.1, low salt), a more structured acid molten globule intermediate I2 (pH 3.3 ± 0.1, 0.42 M NaCl), and the TFE state (pH 3.3 ± 0.1, 42% TFE). It has been found that all denatured states, including GuHCl, TFE, acid denatured and acid molten globule intermediate I1, fold with the same kinetics, provided that the final conditions are identical. This does not apply to the second acid molten globule intermediate I2, which demonstrates a higher rate of folding by a factor of 270. Different energy of activation and pH dependence were found for folding from states I1 or I2. Proteins 32:304-313, 1998. © 1998 Wiley-Liss, Inc.
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  • 17
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    Keywords: cysteine proteinase inhibitors ; cystatins ; human stefin B ; kinetics ; protein folding ; stopped-flow CD ; trifluoroethanol ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The folding of human stefin B has been studied by several spectroscopic probes. Stopped-flow traces obtained by circular dichroism in the near and far UV, by tyrosine fluorescence, and by extrinsic probe ANS fluorescence are compared. Most (60 ± 5%) of the native signal in the far UV circular dichroism (CD) appeared within 10 ms in an unresolved “burst” phase, which was followed by a fast phase (t = 83 ms) and a slow phase (t = 25 s) with amplitudes of 30% and 10%, respectively. Similar fast and slow phases were also evident in the near UV CD, ANS fluorescence, and tyrosine fluorescence. By contrast, human stefin A, which has a very similar structure, exhibited only one kinetic phase of folding (t = 6 s) detected by all the spectroscopic probes, which occurred subsequent to an initial “burst” phase observed by far UV CD. It is interesting that despite close structural similarity of both homologues they fold differently, and that the less stable human stefin B folds faster by an order of magnitude (comparing the non-proline limited phase). To gain more information on the stefin B folding mechanism, effects of pH and trifluoroethanol (TFE) on the fast and slow phases were investigated by several spectroscopic probes. If folding was performed in the presence of 7% of TFE, rate acceleration and difference in the mechanism were observed. Protein 32:296-303, 1998. © 1998 Wiley-Liss, Inc.
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  • 18
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    Keywords: chemical modification ; fluorescent probe ; site-directed mutagenesis ; cysteine-free protein ; alanine scanning ; enzyme reconstitution ; protein-DNA interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A monomercury derivative of fluoresceine acetate (FMMA) was previously suggested as a specific reagent reacting with only one of four cysteine (Cys) residues in the α subunit of Escherichia coli RNA polymerase. Here, we analyzed the reactivity against FMMA of both isolated α subunit and α subunit assembled in the holoenzyme. In both cases, the highest reactivity was identified for Cys-269 positioned in the regulatory helix of C-terminal domain (CTD) which includes the contact sites for both class-I transcription factors and DNA UP elements. Substitution of Ala for both Cys-269 and Cys-176 completely eliminates the reactivity of α subunit against the fluorescent dye, supporting the prediction that another reactive amino acid under native conformation is Cys-176, which is positioned within or near the region important for α dimerization and its binding of β' subunit. In the isolated α subunit, the reactivity against FMMA is different between these two Cys residues and the order is from Cys-269 to Cys-176. Mutant α-subunits, bearing only one Cys residue at either 269 or 176, could be reconstituted into locally modified and active enzymes. This FMMA modification system may provide a tool suitable for studies of intra- and intermolecular interactions of this subunit. Proteins 30:183-192, 1998. © 1998 Wiley-Liss, Inc.
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  • 19
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    Keywords: secondary structure arrangements ; protein structure database ; left/right topology ; knowledge-based structure prediction ; intrinsic stability ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α-β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α-β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193-212, 1998. © 1998 Wiley-Liss, Inc.
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  • 20
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    Keywords: molecular dynamics ; protein dynamics ; computer simulation ; Monte Carlo ; Brownian dynamics ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We present an algorithm for simulating the long time scale dynamics of proteins and other macromolecules. Our method applies the concept of multiple time step integration to the diffusive Langevin equation, in which short time scale dynamics are replaced by friction and noise. The macromolecular force field is represented at atomic resolution. Slow motions are modeled by constrained Langevin dynamics with very large time steps, while faster degrees of freedom are kept in local thermal equilibrium. In the limit of a sufficiently large molecule, our algorithm is shown to reduce the CPU time required by two orders of magnitude. We test the algorithm on two systems, alanine dipeptide and bovine pancreatic trypsin inhibitor (BPTI), and find that it accurately calculates a variety of equilibrium and dynamical properties. In the case of BPTI, the CPU time required is reduced by nearly a factor of 60 compared to a conventional, unconstrained Langevin simulation using the same force field. Proteins 30:215-227, 1998. © 1998 Wiley-Liss, Inc.
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  • 21
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    Keywords: protein folding ; local vs. non-local interactions ; secondary structure prediction ; fragment matching algorithms ; PDB ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: One of the most important questions in the protein folding problem is whether secondary structures are formed entirely by local interactions. One way to answer this question is to compare identical subsequences of proteins to see if they have identical structures. Such an exercise would also reveal a lower limit on the number of amino acids needed to form unique secondary structures. In this context, we have searched the April 1996 release of the Protein Data Bank for sequentially identical subsequences of proteins and compared their structures. We find that identical octamers can have different conformations. In addition, there are several examples of identical heptamers with different conformations, and the number of identical hexamers with different conformations has increased since the previous PDB releases. These observations imply that secondary structure can be formed entirely by non-local interactions and that an identical match of up to eight amino acids may not imply structural similarity. In addition to the larger context of the protein folding problem, these observations have implications for protein structure prediction methods. Proteins 30:228-231, 1998. © 1998 Wiley-Liss, Inc.
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  • 22
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    Keywords: P1 nuclease ; X-ray crystallography ; substrate recognition ; catalytic mechanism ; thiophosphorylated oligonucleotides ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The reaction mechanism of nuclease P1 from Penicillium citrinum has been investigated using single-stranded dithiophosphorylated di-, tetra-, and hexanucleotides as substrate analogs. The complexes crystallize in tetragonal and orthorhombic space groups and have been solved by molecular replacement. The high resolution structures give a clear picture of base recognition by P1 nuclease at its two nucleotide-binding sites, especially the 1.8 Å structure of a P1-tetranucleotide complex which can be considered a P1-product complex. The observed binding modes are in agreement with a catalytic mechanism where the two closely spaced zinc ions activate the attacking water while the third, more exposed zinc ion stabilizes the leaving 03' oxyanion. Stacking as well as hydrogen bonding interactions with the base 5' to the cleaved phosphodiester bond are important elements of substrate binding and recognition. Modelling of a productive P1-substrate complex based on the solved structures suggests steric hindrance as the likely reason for the resistance of Rp-phosphorothioates and phosphorodithioates. Differences with the highly homologous nuclease S1 from Aspargillus oryzae are discussed. Proteins 32:414-424, 1998. © 1998 Wiley-Liss, Inc.
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  • 23
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    Keywords: protein cavity ; molecular dynamics simulation ; free energy calculation ; particle insertion ; protein hydration ; protein ligand binding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Hydration of protein cavities influences protein stability, dynamics, and function. Protein active sites usually contain water molecules that, upon ligand binding, are either displaced into bulk solvent or retained to mediate protein-ligand interactions. The contribution of water molecules to ligand binding must be accounted for to compute accurate values of binding affinities. This requires estimation of the extent of hydration of the binding site. However, it is often difficult to identify the water molecules involved in the binding process when ligands bind on the surface of a protein. Cytochrome P450cam is, therefore, an ideal model system because its substrate binds in a buried active site, displacing partially disordered solvent, and the protein is well characterized experimentally. We calculated the free energy differences for having five to eight water molecules in the active site cavity of the unliganded enzyme from molecular dynamics simulations by thermodynamic integration employing a three-stage perturbation scheme. The computed free energy differences between the hydration states are small (within 12 kJ mol-1) but distinct. Consistent with the crystallographic determination and studies employing hydrostatic pressure, we calculated that, although ten water molecules could in principle occupy the volume of the active site, occupation by five to six water molecules is thermodynamically most favorable. Proteins 32:381-396, 1998. © 1998 Wiley-Liss, Inc.
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  • 24
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    Keywords: theory of protein folding ; folding funnel ; folding thermodynamics ; folding kinetics ; conformation space ; sequence/structure compatibility ; thermal denaturation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: It is hard to construct theories for the folding of globular proteins because they are large and complicated molecules having enormous numbers of nonnative conformations and having native states that are complicated to describe. Statistical mechanical theories of protein folding are constructed around major simplifying assumptions about the energy as a function of conformation and/or simplifications of the representation of the polypeptide chain, such as one point per residue on a cubic lattice. It is not clear how the results of these theories are affected by their various simplifications. Here we take a very different simplification approach where the chain is accurately represented and the energy of each conformation is calculated by a not unreasonable empirical function. However, the set of amino acid sequences and allowed conformations is so restricted that it becomes computationally feasible to examine them all. Hence we are able to calculate melting curves for thermal denaturation as well as the detailed kinetic pathway of refolding. Such calculations are based on a novel representation of the conformations as points in an abstract 12-dimensional Euclidean conformation space. Fast folding sequences have relatively high melting temperatures, native structures with relatively low energies, small kinetic barriers between local minima, and relatively many conformations in the global energy minimum's watershed. In contrast to other folding theories, these models show no necessary relationship between fast folding and an overall funnel shape to the energy surface, or a large energy gap between the native and the lowest nonnative structure, or the depth of the native energy minimum compared to the roughness of the energy landscape. Proteins 32:425-437, 1998. © 1998 Wiley-Liss, Inc.
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  • 25
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    Keywords: protein stability ; conformational free energy ; structure discrimination ; molecular dynamics ; molecular surface ; continuum solvent model ; continuum dielectric model ; boundary element method ; protein entropy ; quasi-harmonic approximation ; deliberately misfolded protein structures ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new method for calculating the total conformational free energy of proteins in water solvent is presented. The method consists of a relatively brief simulation by molecular dynamics with explicit solvent (ES) molecules to produce a set of microstates of the macroscopic conformation. Conformational energy and entropy are obtained from the simulation, the latter in the quasi-harmonic approximation by analysis of the covariance matrix. The implicit solvent (IS) dielectric continuum model is used to calculate the average solvation free energy as the sum of the free energies of creating the solute-size hydrophobic cavity, of the van der Waals solute-solvent interactions, and of the polarization of water solvent by the solute's charges. The reliability of the solvation free energy depends on a number of factors: the details of arrangement of the protein's charges, especially those near the surface; the definition of the molecular surface; and the method chosen for solving the Poisson equation. Molecular dynamics simulation in explicit solvent relaxes the protein's conformation and allows polar surface groups to assume conformations compatible with interaction with solvent, while averaging of internal energy and solvation free energy tend to enhance the precision. Two recently developed methods - SIMS, for calculation of a smooth invariant molecular surface, and FAMBE, for solution of the Poisson equation via a fast adaptive multigrid boundary element - have been employed. The SIMS and FAMBE programs scale linearly with the number of atoms. SIMS is superior to Connolly's MS (molecular surface) program: it is faster, more accurate, and more stable, and it smooths singularities of the molecular surface. Solvation free energies calculated with these two programs do not depend on molecular position or orientation and are stable along a molecular dynamics trajectory. We have applied this method to calculate the conformational free energy of native and intentionally misfolded globular conformations of proteins (the EMBL set of deliberately misfolded proteins) and have obtained good discrimination in favor of the native conformations in all instances. Proteins 32:399-413, 1998. © 1998 Wiley-Liss, Inc.
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  • 26
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    Keywords: mutant T4 lysozyme ; S-2-amino-3-cyclopentylpropanoic acid ; free energy simulation ; protein stability ; packing interaction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Free energy derivatives, pictorial representation of free energy changes (PROFEC) and free energy perturbation methods were employed to suggest the modifications that may improve the stability of a mutant T4 lysozyme with a S-2-amino-3-cyclopentylpropanoic acid residue (Cpe) at position 133. The free energy derivatives and PROFEC methods were used to locate promising sites where modifications may be introduced. The effects of several candidate modifications on the enzyme's stability were analyzed by the free energy perturbation method. We found that this scheme is able to effectively suggest modifications that may increase the enzyme's stability. The modifications investigated are the introduction of a methyl, a tert-butyl or a trifluoromethyl group at the Cε2 position and a cyclopropyl group between the Cδ2 and Cε2 position on the cyclopentyl ring. The stereochemistry of the introduced groups (in the α or β configurations) was studied. Our calculations predict that the introduction of a methyl group in the α configuration or a cyclopropyl group in the β configuration will increase the stability of the enzyme; the introduction of the two groups in the other configurations and the other modifications will decrease the stability of the enzyme. The results indicate that packing interactions can strongly influence the stability of the enzyme. Proteins 32:438-458, 1998. © 1998 Wiley-Liss, Inc.
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  • 27
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    Keywords: protein assembly ; protein structure ; protein reduced models ; lattice models ; Monte Carlo simulations ; fold prediction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A new, efficient method for the assembly of protein tertiary structure from known, loosely encoded secondary structure restraints and sparse information about exact side chain contacts is proposed and evaluated. The method is based on a new, very simple method for the reduced modeling of protein structure and dynamics, where the protein is described as a lattice chain connecting side chain centers of mass rather than Cαs. The model has implicit built-in multibody correlations that simulate short- and long-range packing preferences, hydrogen bonding cooperativity and a mean force potential describing hydrophobic interactions. Due to the simplicity of the protein representation and definition of the model force field, the Monte Carlo algorithm is at least an order of magnitude faster than previously published Monte Carlo algorithms for structure assembly. In contrast to existing algorithms, the new method requires a smaller number of tertiary restraints for successful fold assembly; on average, one for every seven residues as compared to one for every four residues. For example, for smaller proteins such as the B domain of protein G, the resulting structures have a coordinate root mean square deviation (cRMSD), which is about 3 Å from the experimental structure; for myoglobin, structures whose backbone cRMSD is 4.3 Å are produced, and for a 247-residue TIM barrel, the cRMSD of the resulting folds is about 6 Å. As would be expected, increasing the number of tertiary restraints improves the accuracy of the assembled structures. The reliability and robustness of the new method should enable its routine application in model building protocols based on various (very sparse) experimentally derived structural restraints. Proteins 32:475-494, 1998. © 1998 Wiley-Liss, Inc.
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  • 28
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    Keywords: megakaryocyte growth and development factor ; thrombopoietin ; cytokine ; equilibrium denaturation ; conformation ; protein folding ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The effect of pH and urea on the conformation of recombinant human megakaryocyte growth and development factor (rHuMGDF) was determined by circular dichroism, intrinsic fluorescence spectroscopy, and equilibrium ultracentrifugation. The conformation of rHuMGDF was dependent on pH and urea concentration. Multiple folding forms were evidenced by multiple pH-induced transitions and urea-induced equilibrium transitions that deviated from a simple two-state process. In neutral to alkaline pH, rHuMGDF exists as a monomer, but an acid-induced conformational state self-associates to form a soluble aggregate. A folding intermediate(s) was observed with a more stable secondary structure than tertiary structure and was dependent on the pH of the urea-induced denaturation. The differences in the stabilities of the folding states were most distinct in the pH range of 4.5 to 6.5. The presence of intermediates in the folding pathway of rHuMGDF are similar to findings of previous studies of related growth factors that share a common three-dimensional structure. Proteins 32:495-503, 1998. © 1998 Wiley-Liss, Inc.
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  • 29
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    Keywords: cysteine protease ; zymogens ; inhibition ; caricain ; cathepsin L ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Synthetic peptides corresponding to the proregions of papain-like cysteine proteases have been shown to be good and selective inhibitors of their parental enzymes. The molecular basis for their selectivity, quite remarkable in some cases, is not fully understood. The recent determination of the crystal structures of three distinct papain-like cysteine protease zymogens allows detailed structural comparisons to be made. The reasons for the specificity shown by each proregion toward its cognate enzyme are explained in terms of the three-dimensional structure of the proregion and the interface between the mature enzyme and the proregion. These comparisons reveal that insertion and substitution of amino acids within the proregion cause major rearrangement of sidechains on the enzyme/proregion interface, allowing detailed surface and charge recognition. Proteins 32:504-514, 1998. © 1998 Wiley-Liss, Inc.
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  • 30
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    Keywords: protein inhibitors ; serine proteinases ; protein loop ; canonical conformation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Canonical loops of protein inhibitors of serine proteinases occur in proteins having completely different folds. In this article, conformations of the loops have been analyzed for inhibitors belonging to 10 structurally different families. Using deviation in Cα-Cα distances as a criterion for loop similarity, we found that the P3-P3′ segment defines most properly the length of the loop. When conformational differences among loops of individual inhibitors were compared using root mean square deviation (rmsd) in atomic coordinates for all main chain atoms (Δr method) and rmsd operating in main chain torsion angles (Δt method), differences of up to 2.1 Å and 72.3°, respectively, were observed. Such large values indicate significant conformational differences among individual loops. Nevertheless, the overall geometry of the inhibitor-proteinase interaction is very well preserved, as judged from the similarity of Cα-Cα distances between Cα of catalytic Ser and Cα of P3-P3′ residues in various enzyme-inhibitor complexes. The mode of interaction is very well preserved both in the chymotrypsin and subtilisin families, as distances calculated for subtilisin-inhibitor complexes are almost always within the range of those for chymotrypsin-inhibitor complexes. Complex formation leads to conformational changes of up to 160° for χ1 angle. Side chains of residue P1 and P2′ adopt in different complexes a similar orientation (χ1 angle = -60° and -180°, respectively). To check whether the canonical conformation can be found among non-proteinase-inhibitor Brookhaven Protein Data Bank structures, two selection criteria - the allowed main chain dihedral angles and Cα-Cα distances for the P3-P3′ segment - were applied to all these structures. This procedure detected 132 unique hexapeptide segments in 121 structurally and functionally unrelated proteins. Partial preferences for certain amino acids occurring at particular positions in these hexapeptides could be noted. Further restriction of this set to hexapeptides with a highly exposed P1 residue side chain resulted in 40 segments. The possibility of complexes formation between these segments and serine proteinases was ruled out in molecular modeling due to steric clashes. Several structural features that determine the canonical conformation of the loop both in inhibitors and in other proteins can be distinguished. They include main chain hydrogen bonds both within the P3-P3′ segment and with the scaffold region, P3-P4 and P3′-P4′ hydrophobic interactions, and finally either hydrophobic or polar interactions involving the P1′ residue. Proteins 32:459-474, 1998. © 1998 Wiley-Liss, Inc.
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  • 31
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    Keywords: sugar ; acetamido group ; mimicry ; inhibition ; lysozyme ; CDR loop ; VHH ; heavy-chain immunoglobulin ; solvent accessible surface area ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Whereas antibodies have demonstrated the ability to mimic various compounds, classic heavy/light-chain antibodies may be limited in their applications. First, they tend not to bind enzyme active site clefts. Second, their size and complexity present problems in identifying key elements for binding and in using these elements to produce clinically valuable compounds. We have previously shown how cAb-Lys3, a single variable domain fragment derived from a lysozyme-specific camel antibody naturally lacking light chains, overcomes the first limitation to become the first antibody structure observed penetrating an enzyme active site. We now demonstrate how cAb-Lys3 mimics the oligosaccharide substrate functionally (inhibition constant for lysozyme, 50 nM) and structurally (lysozyme buried surface areas, hydrogen bond partners, and hydrophobic contacts are similar to those seen in sugar-complexed structures). Most striking is the mimicry by the antibody complementary determining region 3 (CDR3) loop, especially Ala104, which mimics the subsite C sugar 2-acetamido group; this group has previously been identified as a key feature in binding lysozyme. Comparative simplicity, high affinity and specificity, potential to reach and interact with active sites, and ability to mimic substrate suggest that camel heavy-chain antibodies present advantages over classic antibodies in the design, production, and application of clinically valuable compounds. Proteins 32:515-522, 1998. © 1998 Wiley-Liss, Inc.
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  • 32
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    Keywords: lipid binding ; lipid transfer protein ; maize ; molecular modeling ; NMR ; X-ray ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The three-dimensional solution structure of maize nonspecific lipid transfer protein (nsLTP) obtained by nuclear magnetic resonance (NMR) is compared to the X-ray structure. Although both structures are very similar, some local structural differences are observed in the first and the fourth helices and in several side-chain conformations. These discrepancies arise partly from intermolecular contacts in the crystal lattice. The main characteristic of nsLTP structures is the presence of an internal hydrophobic cavity whose volume was found to vary from 237 to 513 Å3 without major variations in the 15 solution structures. Comparison of crystal and NMR structures shows the existence of another small hollow at the periphery of the protein containing a water molecule in the X-ray structure, which could play an important structural role. A model of the complexed form of maize nsLTP by α-lysopalmitoylphosphatidylcholine was built by docking the lipid inside the protein cavity of the NMR structure. The main structural feature is a hydrogen bond found also in the X-ray structure of the complex maize nsLTP/palmitate between the hydroxyl of Tyr81 and the carbonyl of the lipid. Comparison of 12 primary sequences of nsLTPs emphasizes that all residues delineating the cavities calculated on solution and X-ray structures are conserved, which suggests that this large cavity is a common feature of all compared plant nsLTPs. Furthermore several conserved basic residues seem to be involved in the stabilization of the protein architecture. Proteins 31:160-171, 1998. © 1998 Wiley-Liss, Inc.
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  • 33
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    Keywords: sequence-to-structure correlation ; contact environment ; contact prediction ; Bayesian classification ; cluster analysis ; nearest-neighbor classification ; decision tree classification ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The identification of correlations between sequence patterns and structural motifs is a prerequisite in the development of protein structure prediction methods. The prediction accuracy indicates whether these correlations are discerned. We present an approach to identify long-range relationships between sequence patterns and structural motifs by varying the granulation of the structure description. Since interaction among residues is a major determinant in protein folding, we consider contact environments formed by two triplets of three sequentially neighboring residues and described by vectors whose components express contact strengths on an atomic level. Through testing various classification schemes, including their resolution and optimizing parameters, discernible relationships between sequences and folds are explored. About ten structural contact states, together with information from noncontacting regions, could improve the accuracy of contact prediction. Proteins 31:172-185, 1998. © 1998 Wiley-Liss, Inc.
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  • 34
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    Keywords: accessibility to internal cavities ; crystallographic thermal factors ; ligand binding ; protein dynamic ; protein structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Protein structures are flexible both in solution and in the solid state. X-ray crystallographically determined thermal factors monitor the flexibility of protein atoms. A method utilizing such factors is proposed to delineate protein regions through which a ligand can exchange between binding site and bulk solvent. It is based on the assumption that thermally excited protein regions are excellent candidates for opening a ligand channel. Computationally simple and inexpensive, the method analyzes directions from which thermal factors can propagate within the protein, resulting in thermal motion paths (TMPs). Applications to engineered T4 lysozymes, where an artificial internal cavity can host hydrophobic molecules, and to sperm whale myoglobins, where the active site is completely buried, yielded results in agreement with other independent structural observations and with previous hypotheses. Further new features could also be suggested. The proposed TMP analysis could aid molecular dynamics simulation studies as well as time-resolved and site-directed mutagenesis experimental studies, especially given its modest computational expense and its direct roots in experimental results based on thermal factors determined in high-resolution crystallographic studies. Proteins 31:201-213,1998. © 1998 Wiley-Liss, Inc.
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  • 35
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    Keywords: protein ; molecular recognition ; signal transduction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recognition of Ras by its downstream target Raf is mediated by a Ras-recognition region in the Ras-binding domain (RBD) of Raf. Residues 78-89 in this region occupy two different conformations in the ensemble of NMR solution structures of the RBD: a fully α-helical one, and one where 87-90 form a type IV β-turn. Molecular dynamics simulations of the RBD in solution were performed to explore the stability of these and other possible conformations of both the wild-type RBD and the R89K mutant, which does not bind Ras. The simulations sample a fully helical conformation for residues 78-89 similar to the NMR helical structures, a conformation where 85-89 form a 310-helical turn, and a conformation where 87-90 form a type I |iB-turn, whose free energies are all within 0.3 kcal/mol of each other. NOE patterns and Hα chemical shifts from the simulations are in reasonable agreement with experiment. The NMR turn structure is calculated to be 3 kcal/mol higher than the three above conformations. In a simulation with the same implicit solvent model used in the NMR structure generation, the turn conformation relaxes into the fully helical conformation, illustrating possible structural artifacts introduced by the implicit solvent model. With the Raf R89K mutant, simulations sample a fully helical and a turn conformation, the turn being 0.9 kcal/mol more stable. Thus, the mutation affects the population of RBD conformations, and this is expected to affect Ras binding. For example, if the fully helical conformation of residues 78-89 is required for binding, its free energy increase in R89K will increase the binding free energy by about 0.6 kcal/mol. Proteins 31:186-200, 1998. © 1998 Wiley-Liss, Inc.
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  • 36
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: β-glucosidases (family 3) ; circular permutation ; β/α-barrel ; “mainly all-β” domain ; double-domain topology ; secondary-structure prediction ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: By predicting the general secondary structure for β-glucosidases (family 3), in conjunction with existing knowledge of the circular permutants present in B. fibrisolvens and R. albus, we were able to find the canonical elements of the secondary structure. The way these elements are linked suggests that there is a double-domain topology made up of a (β/α)8-barrel domain and a “mainly all-β” domain. A number of already known conserved motifs are located within (or near) the C-terminal part of the putative parallel β-strands of the (β/α)8-barrel, which is consistent with what is known about the location of catalytical sites for enzymes that have this domain topology. Within the circular permutants, two β/α units are located at the N-terminal part of the molecule, whereas the other six β/α units are located at the C-terminal end. In this way, the circular permutants can be seen to have a putative discontinuous double-domain topology. Proteins 31:214-223, 1998. © 1998 Wiley-Liss, Inc.
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  • 37
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    ISSN: 0887-3585
    Keywords: protein structure prediction ; supersecondary structure ; genetic algorithm ; solvent accessible surface area ; hydrophobic potential ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: We describe an algorithm to compute native structures of proteins from their primary sequences. The novel aspects of this method are: 1) The hydrophobic potential was set to be proportional to the nonpolar solvent accessible surface. To make computation feasible, we developed a new algorithm to compute the solvent accessible surface areas rapidly. 2) The supersecondary structures of each protein were predicted and used as restraints during the conformation searching processes. This algorithm was applied to five proteins. The overall fold of these proteins can be computed from their sequences, with deviations from crystal structures of 1.48-4.48 Å for Cα atoms. Proteins 31:247-257, 1998. © 1998 Wiley-Liss, Inc.
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  • 38
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: MALDI mass spectrometric peptide mapping ; membrane proteins ; in situ gel digestion ; porin ; permeability transition ; noncovalent complexes ; protein interactions ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Mass spectrometric peptide mapping, particularly by matrix-assisted laser desorption-ionization (MALDI-MS), has recently been shown to be an efficient tool for the primary structure characterization of proteins. In combination with in situ proteolytic digestion of proteins separated by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), mass spectrometric peptide mapping permits identification of proteins from complex mixtures such as cell lysates. In this study we have investigated several ion channel membrane proteins (porins) and their supramolecular assembly in mitochondrial membranes by peptide mapping in solution and upon digestion in the gel matrix. Porins are integral membrane proteins serving as nonspecific diffusion pores or as specific systems for the transport of substrates through bacterial and mitochondrial membranes. The well-characterized porin from Rhodobacter capsulatus (R.c.-porin) has been found to be a native trimeric complex by the crystal structure and was used as a model system in this study. R.c.-porin was characterized by MALDI-MS peptide mapping in solution, and by direct in situ-gel digestion of the trimer. Furthermore, in this study we demonstrate the direct identification of the noncovalent complex between a mitochondrial porin and the adenine nucleotide translocator from rat liver, by MALDI-MS determination of the specific peptides due to both protein sequences in the SDS-PAGE gel band. The combination of native gel electrophoresis and mass spectrometric peptide mapping of the specific gel bands should be developed as a powerful tool for the molecular identification of protein interactions. Proteins Suppl. 2:63-73, 1998. © 1998 Wiley-Liss, Inc.
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  • 39
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    New York, NY : Wiley-Blackwell
    ISSN: 0887-3585
    Keywords: mass spectrometry ; matrix-assisted laser desorption/ionization ; electrospray ; database searching ; gel electrophoresis ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future. The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome. Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era. Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems. We describe current strategies and selected applications in molecular and cell biology. The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins. The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples. Proteins Suppl. 2:74-89, 1998. © 1998 Wiley-Liss, Inc.
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  • 40
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    Journal of Molecular Recognition 11 (1998), S. 2-9 
    ISSN: 0952-3499
    Keywords: poliovirus ; human poliovirus receptor ; immunoglobulin superfamily ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The ability of a virus to attach to a suceptible host cell is of utmost importance for the initiation of viral life cycle. Cell surface proteins called viral receptors mediate the initial steps of virus attachment and uptake. Poliovirus (PV) is one of the most studied animal viruses and its interaction with its cellular receptor, the human poliovirus receptor (hPVR) has been well characterized. This review will present our current understanding of the PV/hPVR interaction at the genetic and biochemical level. In addition, we will also discuss the implicatlions of the PV/hPVR interaction on PV tissue tropism and the evolution of the three PV serotypes. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 41
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    Journal of Molecular Recognition 11 (1998), S. 20-24 
    ISSN: 0952-3499
    Keywords: calmodulin ; citrate ; conformation ; modification ; structure ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Calmodulin (CaM) is a major Ca2+ messenger which, upon Ca2+ activation, binds and activates a number of target enzymes involved in crucial cellular processes. The dependence on Ca2+ ion concentration suggests that CaM activation may be modulated by low-affinity Ca2+ chelators. The effect on CaM structure and function of citrate ion, a Ca2+ chelator commonly found in the cytosol and the mitochondria, was therefore investigated. A series of structural and biochemical methods, including tryptic mapping, immunological recognition by specific monoclonal antibodies, CIDNP-NMR, binding to specific ligands and association with radiolabeled citrate, showed that citrate induces conformational modifications in CaM which affect the shape and activity of the protein. These changes were shown to be associated with the C-terminal lobe of the molecule and involve actual binding of citrate to CaM. Analyzing X-ray structures of several citrate-binding proteins by computerized molecular graphics enabled us to identify a putative citrate-binding site (CBS) on the CaM molecule around residues Arg106-His107. Owing to the tight proximity of this site to the third Ca2+-binding loop of CaM, binding of citrate is presumably translated into changes in Ca2+ binding to site III (and indirectly to site IV). These changes apparently affect the structural and biochemical properties of the conformation-sensitive protein. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 42
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    Journal of Molecular Recognition 11 (1998), S. 32-39 
    ISSN: 0952-3499
    Keywords: serine proteases ; trypsins ; subtilisins ; serine protease inhibitors ; IMAC ; histidine mapping ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The immobilized metal ion affinity (IMA) interaction of different serine proteases, namely porcine and bovine trypsins and BPN' and Carlsberg subtilisins, was studied on Sepharose-IDA-CuII. Both trypsins were resolved into their different subspecies, whereas the subtilisins appeared as only one species. The use of diethyl pyrocarbonate-modified enzymes demonstrated the contribution of histidine(s) as the sole interacting site(s). The use of different peptidic and chemical inhibitors complexed to the enzymes confirmed the contribution of histidine(s) as the interacting site(s) and further resulted in different chromatographic patterns for the free and complexed serine proteases. Comparison of the chromatographic data for each enzyme with the accessible surface area calculation by molecular modelling on the available crystallographic structure allowed us to hypothesize a map of the surface-accessible histidine on each enzyme. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 43
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    Journal of Molecular Recognition 11 (1998), S. 52-57 
    ISSN: 0952-3499
    Keywords: confocal microscopy ; immobilization ; liposomes ; chromatography ; freeze-thawing ; freezing effects ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Biological membranes immobilized in chromatographic gel beads constitute a multifunctional affinity matrix. Membrane protein-solute interactions and drug partitioning into the lipid bilayers can conveniently be studied. By the use of confocal laser-scanning microscopy (CLSM) the distribution of immobilized model membranes in the beads has been visualized for the first time. Freeze-thaw-immobilized liposomes in Superdex 200 gel beads were situated in a thick shell surrounding a liposome-free core. The amount of phospholipids immobilized by freeze-thawing was dependent on the temperature in the cooling bath and the type of test tube used. A bath temperature of -25 °C gave higher immobilization yield than freezing at -75 or -8 °C did. Freeze-thawing in the presence of liposomes did not affect the gel bead shape or the refractive index homogeneity of the agarose network of the beads, as shown by confocal microscopy. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 44
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    Journal of Molecular Recognition 11 (1998), S. 69-74 
    ISSN: 0952-3499
    Keywords: chiral recognition ; molecular imprinting ; hydrogen-bonding ; hydrophobic interactions ; polymer ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecularly imprinted polymers (MIPs) prepared using an amide hydrogen-bonding functional monomer (acrylamide) exhibited efficient enantiomeric recognition properties in both organic and aqueous media in the HPLC mode. The results indicate that the amide functional groups formed strong hydrogen-bonding interactions with the template molecule, and specific recognition sites were created within the polymer matrix during the imprinting process. When Boc-L-Trp was used as the template, an MIP prepared in a polar organic solvent (acetonitrile) using acrylamide as the functional monomer showed better enantiomeric recognition of Boc-Trp than the MIPs prepared in the same solvent using an acidic (methacrylic acid) or a basic (2-vinylpyridine) functional monomer or a combination of an acidic and a basic functional monomer (methacrylic acid + 2-vinylpyridine). Our results indicate that in organic media the degree of retention of the sample molecule on the imprinted polymer was controlled by hydrogen-bonding interactions between the sample molecule and the polymer, while in aqueous media it was determined to a considerable extent by hydrophobic interactions. In both media the shape, size and the nature of the hydrogen-bonding groups of the sample molecules were all important factors in determining the enantiomeric and substrate selectivity. In the aqueous media, however, the hydrophobicity of the sample molecules was also found to play an important role. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 45
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    Journal of Molecular Recognition 11 (1998), S. 83-86 
    ISSN: 0952-3499
    Keywords: dissociation constants ; intermolecular interactions ; molecular imprinting ; molecular recognition ; self-assembly ; template polymerization ; ultraviolet spectroscopy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A method for the rapid estimation of the extent of complex formation in molecular imprinting pre-polymerization mixtures is described. By the use of a UV spectroscopy titration procedure, apparent binding constants for such self-assembly processes have been obtained. This method was used for comparison of the interactions between a dipeptide template (N-acetyl-L-phenylalaninyl-L-tryptophanyl methyl ester) and the functional monomer methacrylic acid, and the monomer analogues acetic acid and trifluoroacetic acid. The importance of template-monomer association during the molecular imprinting pre-polymerization phase is discussed with respect to the systems studied. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 46
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    Journal of Molecular Recognition 11 (1998), S. 94-97 
    ISSN: 0952-3499
    Keywords: amino acid ; cyclodextrin ; enthalphy-entropy compensation ; HPLC ; hydrophobic effect ; molecular imprinting ; molecular recognition ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A novel molecularly imprinted polymer (MIP) system selective for D-phenylalanine is described where polymerization is performed in aqueous solution. The unique polymer system comprises a hydrophobic moiety-selective functional monomer, polymerizable β-cyclodextrin, an electrostatic interacting functional monomer, 2-acryloylamido-2-methylpropane sulfonic acid (AMPSA), and the crosslinking agent N,N′-diacryloylpiperazine. Chromatographic evaluation of polymer-ligand recognition characteristics demonstrated ligand selectivity by the MIP and that optimal recognition was achieved through a balance of hydrophobic and electrostatic ligand-polymer interactions, indicating that recognition in these systems is regulated by enthalpy-entropy compensation. The imprinting effect was shown to be sufficient to reverse the inherent selectivity of cyclodextrin for L-phenylalanine. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 47
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    Journal of Molecular Recognition 11 (1998), S. 103-106 
    ISSN: 0952-3499
    Keywords: crown ether ; HPLC ; host-guest chemistry ; molecular imprinting technology ; molecular recognition ; molecularly imprinted polymers ; supramolecular chemistry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Molecularly imprinted polymers have been prepared against aniline and a bis-aniline compound, making use of a crown ether (18-crown-6) to solubilize the monomer-template complexes. Subsequent chromatographic rebinding studies in the absence of crown ether revealed regioselectivity for the templates in the respective polymers. This study indicates that crown ethers can be potentially useful in conjunction with molecular imprinting to solubilize and imprint organic solvent-insoluble compounds. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 48
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    Journal of Molecular Recognition 11 (1998), S. 117-118 
    ISSN: 0952-3499
    Keywords: Recombinant antibody ; thyroglobuline ; gene therapy ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Recombinant antibodies and antibody fragments are currently being produced. They can be used in vitro for the structural study of antigen-antibody interactions for instance, but their in vivo production may have applications for gene therapy of certain cancers and severe viral diseases and in developing new animal models of autoimmune disease. We report here these two types of applications using a recombinant anti-human thyroglobulin (hTg) antibody. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 49
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    Journal of Molecular Recognition 11 (1998), S. 121-125 
    ISSN: 0952-3499
    Keywords: phage display ; biopanning ; affinity chromatography ; bioinformatics ; peptide ; peptidomimetics ; protein A ; immunoglobulin G ; binding domain ; molecular modeling ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The pFc′ fragments of a humanized IgG1 monoclonal antibody were generated by digestion with immobilized pepsin. These pFc′ fragments were separated from F(ab′)2 fragments by affinity chromatography. The pFc′ fragments corresponding to the constant region of the humanized IgG1 monoclonal antibody were used as targets for phage display using variable-length peptide libraries. Interacting phage-displayed peptides were selected by repetitious cycles of target screening and phage amplification. Peptide sequences, deduced by sequencing DNA from isolated phage, were aligned and analyzed for amino acid motifs against each other and protein A. These results indicated that an amino acid motif has been identified using phage display technology that is sufficient for pFc′ binding. Furthermore, the peptides derived from this study may prove useful in the development of peptidomimetic alternatives to protein A for use in affinity chromatography. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 50
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    Journal of Molecular Recognition 11 (1998), S. 128-133 
    ISSN: 0952-3499
    Keywords: immunoglobulin purification ; combinatorial chemistry ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318-IgG interaction was 0.3 µM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS-PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg-1 while intravenous toxicity was close to 150 mg kg-1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 51
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    Journal of Molecular Recognition 11 (1998), S. 175-177 
    ISSN: 0952-3499
    Keywords: immunosensor ; capacitance ; antibody-antigen affinity interactions ; Si-SiO2-Si3N4. ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: There is widespread interest in capacitance immunosensor systems which directly detect antigen binding to immobilized antibody. Our system comprises an active biolayer of antibodies bound to a silicon-silicon dioxide-silicon nitride (Si-SiO2-Si3N4) surface. As with other groups, our system initially gave poorly reproducible responses on addition of antigen. We mechanically degraded the Si-SiO2-Si3N4 surface, and the responses on addition of transferrin were monitored. The mechanical degradation allowed the affinity reaction to be ‘seen’ capacitively. Once the system was established, a comparison of capture antibodies was performed to establish the most effective biolayer. Three affinity reactions were examined: (a) 1D2A4, monoclonal antibody (mAb) to human transferrin, as the capture layer; (b) polyclonal goat anti-human transferrin antibody (PcAb) as the capture layer; and (c) 1D2A4 with transferrin (Tf) prebound as the capture layer. There was no response to addition of transferrin where 1D2A4 was the capture layer. Addition of transferrin when the polyclonal antibody was used as the primary layer resulted in a drop in measured capacitance. Addition of goat anti-human transferrin antibody to a device with 1D2A4 plus transferrin as the capture layer also resulted in a measured capacitance decrease. There is a difference in dielectric/blocking effectiveness between the monoclonal and polyclonal antibodies. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 52
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    Journal of Molecular Recognition 11 (1998), S. 188-190 
    ISSN: 0952-3499
    Keywords: weak affinity ; surface plasmon resonance ; carbohydrate ; antibody-antigen interactions ; hook effect ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Interactions between the immobilized weak-affinity monoclonal IgG antibody 39.5, which is specific for the glucose-α1,4-glucose motif, and various oligosaccharides were studied with surface plasmon resonance technology. The antibody was immobilized at high levels on the surface of the sensor chip and different concentrations of the analytes were injected at 25 and 40 °C. The 39.5 antibody exhibited specific binding to maltose, tetraglucose and maltotriose, with dissociation constants Kd in the range from 0.07 mM (25 °C) to 1.0 mM (40 °C). Association and dissociation rate constants (ka and kd) were rapid and baseline was obtained almost immediately after the end of each antigen injection. This excluded the need for a regeneration step but also made calculation of the kinetic values impossible. Owing to the weak affinity and the small size of the analytes (〈1000 Da), a careful design of control surfaces is demanded to exclude artefactual results. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 53
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    Journal of Molecular Recognition 11 (1998), S. 200-203 
    ISSN: 0952-3499
    Keywords: antibody fragment ; scFv ; immobilization ; biosensor ; binding site ; scanning electron microscopy ; CM5 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N-hydroxysuccinimide (NHS) and N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite™ from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti-IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 54
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    Journal of Molecular Recognition 11 (1998), S. 217-221 
    ISSN: 0952-3499
    Keywords: purification ; ligands ; expanded bed adsorption ; affinity separations ; cell separations ; nucleic acids ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The potential for the use of affinity ligands in expanded bed adsorption (EBA) procedures is reviewed. The use of affinity ligands in EBA may improve its use in direct recovery operations, as the enhanced selectivity of the adsorbent permits selective capture of the target from complex feedstocks and high degrees of purification. The properties of ligands suitable for use in EBA processes are identified and illustrated with examples. In addition to its use in the recovery of soluble products, such as proteins and nucleic acids, from particulate feedstocks, EBA can also be used to recover particulate entities, such as cells and packaged DNA (viruses and phages), from feedstocks. Affinity ligands coupled to appropriate chosen support materials will be required for such processes in order to achieve the necessary selectivity for the required particulate entity. The latter point is illustrated by the use of proteinaceous ligands immobilized to perfluorocarbon emulsions to achieve separations of microbial cells. Copyright © 1998 John Wiley & Sons, Ltd.
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  • 55
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    Journal of Molecular Recognition 11 (1998), S. 10-13 
    ISSN: 0952-3499
    Keywords: antibody-antigen bond ; thermodynamics ; affinity maturation ; calorimetry ; lysozyme ; D1.3 ; scFv ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: Understanding the structural and dynamic determinants of binding free energy in the antigen-antibody bond is of great interest. Much work has focused on selective mutations in order to locate key interaction residues, but this generally results in reduced affinity. The present work instead examines a higher-affinity mutant to characterize the thermodynamic pathway of the affinity maturation process. We have compared the antigen binding energetics of scFv D1.3, an anti-hen egg lysozyme single chain antibody, with a higher-affinity mutant (Hawkins, R. E., Russell, S. J., Baier, M. and Winter, G. (1993). J. Mol. Biol. 234, 958-964). The mutant has fivefold higher affinity for lysozyme but nearly the same enthalpy and heat capacity change upon binding, as measured by isothermal titration calorimetry. Thus, much of the binding free energy difference can be attributed to entropic effects. Fluorescence quenching with acrylamide indicates that this more favorable entropy change may result from a more flexible mutant-lysozyme complex and thus be a configurational entropy effect. Copyright © 1998 John Wiley & Sons, Ltd.
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    Journal of Molecular Recognition 11 (1998), S. 25-27 
    ISSN: 0952-3499
    Keywords: artificial chaperone ; polyelectrolyte complexes ; glyceraldehyde-3-phosphate dehydrogenase ; enzyme inactivation ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: The simplified model of chaperone action when the inactive misfolded forms are removed from the reaction media preventing aggregation was developed using antibodies in combination with polyelectrolyte complexes. The antibodies, which bind specifically inactive dimers of glyceraldehyde-3-phosphate dehydrogenase but not native tetramers, were coupled covalently to poly(methacrylic acid). The treatment of inactivated GAPDH with this conjugate followed by its precipitation after equimolar addition of polycation, poly-(N-ethyl-4-vinylpyridinium bromide), resulted in a significant increase in the specific activity of the enzyme. Copyright © 1998 John Wiley & Sons, Ltd
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    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 49-51 
    ISSN: 0952-3499
    Keywords: human rhinovirus ; receptor ; infection inhibition ; low-density lipoprotein receptor ; baculovirus ; Sf9 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: A fragment of the human low-density lipoprotein receptor encompassing the seven ligand-binding repeats fused to a C-terminal oligo-His tag was expressed in Sf9 insect cells. The melittin signal sequence encoded in the baculovirus vector led to secretion of the protein into the cell supernatant in a soluble form. The receptor fragment bound its natural ligand β-migrating very-low-density lipoprotein and human rhinovirus serotype 2 in non-reducing ligand blots. Infection of all minor group human rhinovirus serotypes investigated was inhibited by the presence of the receptor fragment during viral challenge of HeLa cells. Infection is inhibited by aggregation of the virions. Copyright © 1998 John Wiley & Sons, Ltd.
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