Biophysical Characterization of Proteins in the Post-genomic Era of Proteomics

摘要

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. 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. The genomic era of biomedical research in the 1990s provided massive amounts of information on DNA sequences from many species culminating in the nearly completed human genome sequence (1.Lander E.S. Linton L.M. Birren B. Nusbaum C. Zody M.C. Baldwin J. Devon K. Dewar K. Doyle M. FitzHugh W. Funke R. Gage D. Harris K. Heaford A. Howland J. et al.Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Google Scholar, 2.Venter J.C. Adams M.D. Myers E.W. Li P.W. Mural R.J. Sutton G.G. Smith H.O. Yandell M. Evans C.A. Holt R.A. Gocayne J.D. Amanatides P. Ballew R.M. Huson D.H. Wortman J.R. et al.The sequence of the human genome.Science. 2001; 291: 1304-1351Google Scholar). This wealth of data has been annotated and is continuing to be analyzed by new bioinformatic algorithms. Many protein sequences have been inferred from open reading frames from the more than 50 complete genome sequences (www.tigr.org/tdb/tgi) that include human, rodents, bacteria, viruses, and plants. The next two experimental steps are well underway in the 21st century. The aim of proteomics is to determine the structure, function, and expression of all proteins and their isoforms of a genome. The aim of structural genomics is to clone, express, and determine the three-dimensional structures of many proteins by high throughput x-ray or NMR analyses with the added benefit of defining all folding motifs and, in some cases, function of