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Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods 总被引:9,自引:0,他引:9
Day YS Baird CL Rich RL Myszka DG 《Protein science : a publication of the Protein Society》2002,11(5):1017-1025
The binding interactions of small molecules with carbonic anhydrase II were used as model systems to compare the reaction constants determined from surface- and solution-based biophysical methods. Interaction data were collected for two arylsulfonamide compounds, 4-carboxybenzenesulfonamide (CBS) and 5-dimethyl-amino-1-naphthalene-sulfonamide (DNSA), binding to the enzyme using surface plasmon resonance, isothermal titration calorimetry, and stopped-flow fluorescence. We demonstrate that when the surface plasmon resonance biosensor experiments are performed with care, the equilibrium, thermodynamic, and kinetic constants determined from this surface-based technique match those acquired in solution. These results validate the use of biosensor technology to collect reliable data on small molecules binding to immobilized macromolecular targets. Binding kinetics were shown to provide more detailed information about complex formation than equilibrium constants alone. For example, although carbonic anhydrase II bound DNSA with twofold higher affinity than CBS, kinetic analysis revealed that CBS had a fourfold slower dissociation rate. Analysis of the binding and transition state thermodynamics also revealed significant differences in the enthalpy and entropy of complex formation. The lack of labeling requirements, high information content, and high throughput of surface plasmon resonance biosensors will make this technology an important tool for characterizing the interactions of small molecules with enzymes and receptors. 相似文献
84.
Developmental programming: State‐of‐the‐science and future directions–Summary from a Pennington Biomedical symposium 下载免费PDF全文
85.
William J. Sutherland Rosalind Aveling Thomas M. Brooks Mick Clout Lynn V. Dicks Liz Fellman Erica Fleishman David W. Gibbons Brandon Keim Fiona Lickorish Kathryn A. Monk Diana Mortimer Lloyd S. Peck Jules Pretty Johan Rockström Jon Paul Rodríguez Rebecca K. Smith Mark D. Spalding Femke H. Tonneijck Andrew R. Watkinson 《Trends in ecology & evolution》2014
86.
Dinesh C. Soares Paul N. Barlow David J. Porteous Rebecca S. Devon 《Journal of molecular modeling》2009,15(2):113-122
Defects in the human ALS2 gene, which encodes the 1,657-amino-acid residue protein alsin, are linked to several related motor neuron diseases. We created
a structural model for the N-terminal 690-residue region of alsin through comparative modelling based on regulator of chromosome
condensation 1 (RCC1). We propose that this alsin region contains seven RCC1-like repeats in a seven-bladed beta-propeller
structure. The propeller is formed by a double clasp arrangement containing two segments (residues 1–218 and residues 525–690).
The 306-residue insert region, predicted to lie within blade 5 and to be largely disordered, is poorly conserved across species.
Surface patches of evolutionary conservation probably indicate locations of binding sites. Both disease-causing missense mutations—Cys157Tyr
and Gly540Glu—are buried in the propeller and likely to be structurally disruptive. This study aids design of experimental
studies by highlighting the importance of construct length, will enhance interpretation of protein–protein interactions, and
enable rational site-directed mutagenesis.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
87.
Dale G. Nimmo Sarah Avitabile Sam C. Banks Rebecca Bliege Bird Kate Callister Michael F. Clarke Chris R. Dickman Tim S. Doherty Don A. Driscoll Aaron C. Greenville Angie Haslem Luke T. Kelly Sally A. Kenny Jos J. Lahoz‐Monfort Connie Lee Steven Leonard Harry Moore Thomas M. Newsome Catherine L. Parr Euan G. Ritchie Kathryn Schneider James M. Turner Simon Watson Martin Westbrooke Mike Wouters Matthew White Andrew F. Bennett 《Biological reviews of the Cambridge Philosophical Society》2019,94(3):981-998
Movement is a trait of fundamental importance in ecosystems subject to frequent disturbances, such as fire‐prone ecosystems. Despite this, the role of movement in facilitating responses to fire has received little attention. Herein, we consider how animal movement interacts with fire history to shape species distributions. We consider how fire affects movement between habitat patches of differing fire histories that occur across a range of spatial and temporal scales, from daily foraging bouts to infrequent dispersal events, and annual migrations. We review animal movements in response to the immediate and abrupt impacts of fire, and the longer‐term successional changes that fires set in train. We discuss how the novel threats of altered fire regimes, landscape fragmentation, and invasive species result in suboptimal movements that drive populations downwards. We then outline the types of data needed to study animal movements in relation to fire and novel threats, to hasten the integration of movement ecology and fire ecology. We conclude by outlining a research agenda for the integration of movement ecology and fire ecology by identifying key research questions that emerge from our synthesis of animal movements in fire‐prone ecosystems. 相似文献
88.
The protozoan pathogens Giardia lamblia and Cryptosporidium parvum are major causes of waterborne enteric disease throughout the world. Improved detection methods that are very sensitive and rapid are urgently needed. This is especially the case for analysis of environmental water samples in which the densities of Giardia and Cryptosporidium are very low. Primers and TaqMan probes based on the beta-giardin gene of G. lamblia and the COWP gene of C. parvum were developed and used to detect DNA concentrations over a range of 7 orders of magnitude. It was possible to detect DNA to the equivalent of a single cyst of G. lamblia and one oocyst of C. parvum. A multiplex real-time PCR (qPCR) assay for simultaneous detection of G. lamblia and C. parvum resulted in comparable levels of detection. Comparison of DNA extraction methodologies to maximize DNA yield from cysts and oocysts determined that a combination of freeze-thaw, sonication, and purification using the DNeasy kit (Qiagen) provided a highly efficient method. Sampling of four environmental water bodies revealed variation in qPCR inhibitors in 2-liter concentrates. A methodology for dealing with qPCR inhibitors that involved the use of Chelex 100 and PVP 360 was developed. It was possible to detect and quantify G. lamblia in sewage using qPCR when applying the procedure for extraction of DNA from 1-liter sewage samples. Numbers obtained from the qPCR assay were comparable to those obtained with immunofluorescence microscopy. The qPCR analysis revealed both assemblage A and assemblage B genotypes of G. lamblia in the sewage. No Cryptosporidium was detected in these samples by either method. 相似文献
89.