Abstract
Proteomics focuses on the high throughput study of the expression, structure, interactions, and, to some extent, function of large numbers of proteins. A true understanding of the functioning of a living cell also requires a quantitative description of the stoichiometry, kinetics, and energetics of each protein complex in a cellular pathway. Classical molecular biophysical studies contribute to understanding of these detailed properties of proteins on a smaller scale than does proteomics in that individual proteins are usually studied. This perspective article deals with the role of biophysical methods in the study of proteins in the proteomic era. Several important physical biochemical methods are discussed briefly and critiqued from the standpoint of information content and data acquisition. The focus is on conformational changes and macromolecular assembly, the utility of dynamic and static structural data, and the necessity to combine experimental approaches to obtain a full functional description. The conclusions are that biophysical information on proteins is a useful adjunct to "standard" proteomic methods, that data can be obtained by high throughput technology in some instances, but that hypothesis-driven experimentation may frequently be required.
Highlights
Proteomics focuses on the high throughput study of the expression, structure, interactions, and, to some extent, function of large numbers of proteins
A proteomic approach may focus on limited aspects of this broad menu of protein attributes or, unlike a genomic approach, may focus on a more limited proteome from cell fractionation such as that complement related to a cellular organelle or component
Identification of the ground rules that govern function gets bypassed in the rush to accumulate high throughput protein structures or to identify interactomes; many investigators do emphasize the importance of a quantitative assessment of function
Summary
Conformations at low resolution and changes in conformation with different conditions have, traditionally, been studied by spectroscopic methods that are highly sensitive and rapid. The rapidity or instantaneous measurement of the signal allows kinetics of the processes under study to be measured and suggests that high throughput analyses could be developed, if desirable These classical and neo-classical methods include ultraviolet light absorption difference spectroscopy, intrinsic fluorescence, extrinsic fluorescence with covalent or noncovalent probes, fluorescence resonance energy transfer in many variations, CD, and Fourier transform infra-red spectroscopy. Binding of 1-anilino-8-naphthalenesulfonate, or a similar fluorophore, to hydrophobic protein surfaces is often taken to indicate partially unfolded molten globule regions of proteins [12] Such methods could be automated to provide a high throughput survey of a family. A situation might be delineated in which such an analysis would be warranted but would represent a limited set of all proteins, e.g. pathological or neurological
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