Biophysical methods

In practice, measurement of the individual rate constants or equilibrium constants for these various chemical steps requires specialized methodologies, such as transient state kinetics (see Johnson, 1992, Copeland, 2000, and Fersht, 1999, for discussion of such methods) and/or a variety of biophysical methods for measuring equilibrium binding (Copeland, 2000). These specialized methods are beyond the scope of the present text. More commonly, the overall rate of reaction progress after ES complex formation is quantified experimentally in terms of a composite rate constant given the symbol km. 

The equilibrium population is said to have a structure when a substantial fraction of the molecules adopts similar conformations. But the phrase lacking structure does not imply that individual molecules comprising the ensemble lack a conformation rather, the population is too heterogeneous to be readily characterized using a coherent, structure-based descriptor. The unfolded state resists ready characterization because it is so diverse. Typical biophysical methods report ensemble-... 

Plaxco, K. W., and Dobson, C. M. (1996). Time-resolved biophysical methods in the study of protein folding. Curr. Opin. Strud. Biol. 6, 630-636. 

Volume 430. Translation Initiation Reconstituted Systems and Biophysical Methods... 

Shchipunov, Yu.A., Karpenko, T.Yu., Bakunina, I.Yu., Burtseva, Yu.V. and Zvyagintseva, T.N. (2004) A new precursor for the immobilization of enzymes inside sol-gel derived hybrid silica nanocomposites containing polysaccharides. Journal of Biochemical and Biophysical Methods, 58, 25-38. 

Cross-linking constrains the conformational flexibility of biopolymers and, as a rule, stabilizes their secondary, tertiary, and quaternary structures against the denaturing effects of high temperatures.29 We used differential scanning calorimetry (DSC) to compare the heat-induced conformational transitions of selected RNase A samples that were characterized in Figure 15.2. A brief introduction to DSC is provided in Section 15.15.1 for those readers unfamiliar with this biophysical method. Trace 1 in Figure 15.3a is the heat absorption... 

The effect of formalin-treatment on the structural properties of RNase A was examined using circular dichroism (CD) spectropolarimetry. A brief introduction to CD spectropolarimetry is provided in Section 15.15.2 for those readers unfamiliar with this biophysical method. The secondary structure of RNase A consists of one long four-stranded anti-parallel p-sheet and three short a-helixes,44 which places RNase A in the a + p structural class of proteins. The effect of a 9-day incubation of RNase A (6.5mg/mL) in 10% formalin on the protein secondary structure was examined with CD spectropolarimetry in the far-UV region (170-240nm) as shown in Figure 15.6a. The resulting... 

Nevertheless most of the biophysical methods do not observe single molecules but a huge amount of players in a concentration range between 1014 and 1020 molecules/1 (corresponding to 100 pmol/1 to 1 mmol/1, which are reasonable concentrations in cellular systems). While the dynamics of the single reactants is stochastic the macroscopic readout of the measuring system normally can be described with ordinary differential equations. 

Here biophysical methods contributed a reductionistic approach. By analyzing a limited number of actors under well defined conditions, the mechanisms of nucleotide exchange, intrinsic and stimulated GTP-hydrolysis, effector binding, and membrane attachment have been elaborated to present a comprehensive model. 

In future, two developments may be expected. On the one hand, elaborated biophysical methods will measure interactions in the cellular context and dissect complex biological patterns. On the other hand, established physico-chemical techniques mainly based on fluorescence will be utilized in high throughput screens to investigate new substances upon their interference with Ras function. 

Glasel JA, Deutscher MP (1995) Introduction to biophysical methods for protein and nucleic acid research. Academic Press, San Diego (ISBN 0-12-286230-9)... 

Garfin, D.E., in Introduction to Biophysical Methods for Protein and Nucleic Acid Research (Eds. J.A. Glasel and M.P. Deutscher), p. 53, Academic Press, San Diego, 1995. 

The costs associated with hit identification using diverse assay systems and technologies as well as, alternatively, biophysical methods and approaches are regarded... 

Boehm, H.J., Boehringer, M., Bur, D., Gmuender, H., Huber, W., Klaus, W., Kostrewa, D., Kuehne, H., Luebbers, T., Meunier-Keller, N., and Mueller, F. Novel inhibitors of DNAgyrase 3D structure based biased needle screening, hit validation by biophysical methods,... 

Day, Y.S., Baird, C.L., Rich, R.L, Myszka, D.G. Direct comparison of binding equilibrium, thermodynamic, and rate constants determined by surface- and solution-based biophysical methods. Protein Sci. 

Braisted, A.C., Hyde, J., McDowell, R.S., Randal, M., Waal, N.D., Wilkinson, J., Yu, C.H., Arkin, M.R. Integrating fragment assembly and biophysical methods in the chemical advancement of small-molecule antagonists of IL-2 an approach for inhibiting protein-protein... 

NMR spectroscopy and X-ray crystallography are the two most common biophysical methods nsed in the determination of high-resolntion strnctnres of macro-molecnles. Each method has inherent strengths and weakness dne to the natnre of... 

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