Department of Biochemistry and Molecular Biology, Finch University of Health Sciences/Chicago Medical School, North Chicago, Illinois 60064
¶ Department of Human Biological Chemistry and Genetics, The University of Texas Medical Branch, Galveston, Texas 77555-1055
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ABSTRACT |
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GENOMICS, PROTEOMICS, AND PHYSICAL BIOCHEMISTRY |
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Proteomics may be defined broadly as the study of all proteins (and alternatively spliced varieties) expressed by a genome, including the concomitant isolation, identification, structural determination (with post-translational modifications), interaction with partners (other proteins, lipids, nucleic acids), expression, developmental time courses, affects on biological responses, and functional properties. Implicit in this definition is the utilization of high throughput methods that approach the actual concomitant accumulation of these data. In practice, a proteomic approach may focus on limited aspects (e.g. identification and interactions) 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 (e.g. mitochondria) or component (e.g. nucleosome). Frequently, identification of the ground rules that govern function gets bypassed in the rush to accumulate high throughput protein structures or to identify interactomes; however, many investigators do emphasize the importance of a quantitative assessment of function. Implied in much of the high throughput approach is that discovery-based research will surpass hypothesis-driven experiments as a means of scientific progress, again a point for contention.
Proteomics has focused primarily on technology involving separation (two-dimensional gels, liquid chromatography, mass spectrometry), structure (x-ray and NMR), and interactions (mass spectrometry, yeast two-hybrid, immunoprecipitations, combinatorial methods) and a shift toward studying hundreds or thousands of proteins at a time. A truly erudite, comprehensive understanding of the functioning of a living cell also requires a quantitative, dynamic description of the stoichiometry, the kinetics of formation, the energetics of the formation, and the functional consequences of each protein complex in a cellular pathway. Classical biophysical analysis to determine size, shape, and solution properties of proteins has been around since the time of viscosity measurements and sedimentation analysis in the analytical centrifuge (3). Biophysical studies in the first half of the 20th century gave birth to our concept of proteins as defined globular or fibrous proteins of exact composition that were amenable to a more refined structural analysis at the atomic level. We are now, perhaps, past puberty and into the adolescent years of growth, some years before a mature understanding of everything there is to know about the full complement of proteins in an organism. From a purely structural viewpoint, maturity is well advanced. The question that will be dealt with in this article will be, "What is the role of biophysical methods in the study of proteins in the proteomic era?" The issues will deal with the information content of physical biochemical methods, dynamic versus static structural data, and interactions in solution.
Biophysical approaches can provide data, complementary to the detailed molecular protein structures from crystallography and high resolution NMR, that reveal insights into how proteins behave in solution and how they interact dynamically with each other. Useful techniques include hydrodynamic methods (analytical ultracentrifugation, viscometry, etc.), thermodynamic methods (light scattering, microcalorimetry, surface plasma resonance), and spectroscopy (fluorescence, circular dichroism (CD),1 electron paramagnetism). In this article we will discuss the proteomic approach of these techniques with experimental examples.
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CONFORMATIONAL CHANGES IN SOLUTION |
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The advantage that spectroscopic methods possess is the ability to vary conditions. The knowledge of the kinetics of a conformational change is important in interpreting the role of the transition in a biological process. Furthermore, knowledge of the G of activation (
G*) from the kinetics often provides an understanding of the transition state and kinetic pathway for the conformational process. A measurement of
G,
H, and
S from temperature dependence of a conformational change establishes the energetic relationship between the forms and, therefore, the ability of external conditions to be able to shift between isomeric forms. From x-ray or NMR studies one may get two or three atomic resolution three-dimensional structures with different ligands bound; from solution spectroscopy one may get a continuous variation of distribution of structures between several conditions, e.g. ligand binding, pH, or temperature.
Stability
One particular variation of this procedure is the measurement of the stability of a native structure by measuring the G for unfolding by temperature or denaturant, as monitored by spectroscopy (4). Would it be possible to automate the procedure for
G of stability determination via high throughput? Probably. Would it be useful to have the
G of stability determined for as many proteins as possible? Maybe. First, automation of a spectrophotometer/fluorometer that has been programmed to increase temperature or add a protein pre-incubated with denaturant would be quite feasible in the modern electronic/robotic era. The feasibility would be dependent on the cost and the demand for such large-scale measurements. Second, thermodynamic stabilities of more than a hundred different proteins have already been determined. General theories have been made based on these measurements (5), and more values would be helpful to extend such theories. However, because the general principles of folding and stability are fairly well understood (6), accumulation of more numbers may not provide more general insights. In this case, the understanding of individual proteins may benefit from
G stability measurements but should probably be done on a case-by-case basis or in a closely related family such as T7 lysozyme or gene V protein (7, 8) with a series of mutations to compare changes in stability.
A good example of a potential high throughput spectroscopic method to scan many proteins rapidly is the use of Congo Red (or thioflavine S) binding, and particularly the birefringence of binding, to determine the presence of amyloid structure in proteins (9). Especially relevant to this discussion is the use of Congo Red binding, along with fluorescence and CD, with those proteins that undergo a reversible transition between an amyloid form and a non-amyloid form, e.g. the prions (10, 11). Similarly, 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). Indeed, such methods could be automated to provide a high throughput survey of a family of proteins for amyloid- or molten globule-forming transitions subsequent to separation by high pressure liquid chromatography or other methods. 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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MACROMOLECULAR ASSEMBLY |
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Transport Techniques
The size and shape of molecules can be estimated by monitoring the movement of these molecules in a force field such as centrifugal force for sedimentation, gravity for gel filtration, and electrical potential for electrophoresis. The principles that govern these techniques are the same, and thus the information content derived from these techniques is similar. The choice of a specific technique depends on the system that is under investigation, e.g. stability, availability of material, and equilibrium constant of the reaction. These approaches provide the information to monitor macromolecular assembly processes. These classical approaches are based on rigorous theoretical principles but have fallen from favor mainly because of the difficulties in using the instruments, the relative slowness of data acquisition, and the complexity of analyzing the data. However, with the recent advent of the new generation of instruments with state-of-the-art technology, these methods have become user-friendly. Furthermore, the availability of powerful software has enabled the users to extract the quantitative parameters with relative ease and speed. The reaction boundary contains data to provide information on the stoichiometry and equilibrium constant of macromolecular complex formation (18). This approach has been applied successfully to monitor the association between different transcription factors Smad 3 and Smad 4 (19), to determine assembly mechanisms of kinesin motor domains (20), to detect the presence of heterogeneity of assembly in ß-lactoglobulin because of genetic variance (21), and to detect isomerization of conformational states in aspartyl transcarbamylase (22).
Gel filtration, particularly the large zone approach, provides the same information as the reaction boundary in sedimentation (2325) but has an added advantage in its flexibility in methods of detection of the elution profile. Fluorescence, radioactivity, or enzyme activity may be used to monitor the elution of samples. Thus, gel filtration under the appropriate circumstances can extend the capability to monitor dissociation constants accurately to values as low as 10-10 M (26).
Light Scattering
Light scattering has undergone a period of renaissance as a result of new advances in instrumentation and development of new software. Current solution filters are very efficient in eliminating the chronic problem of dust particles. With the availability of a large number of interesting biological systems, this approach should again be considered seriously for studying macromolecular assembly, in particular, the kinetics of assembly processes (27, 28). A potential advantage of this approach is the distinct possibility of automation leading to a high throughput type of analysis. Dynamic or quasielastic light scattering technology with automated data analysis has improved in recent years (29) and provides a complement to static light scattering for determining radius of gyration and diffusion coefficient, as well as molecular mass (see below). Dynamic light scattering is currently used extensively to determine homogeneity and, hence, the capability of the protein to crystallize.
Fluorescence Anisotropy
Interaction between macromolecules can be detected by monitoring the changes in the steady state fluorescence anisotropy of a fluorescently labeled species. Basically, fluorescence anisotropy is a measure of the tumbling motion of the fluorescent probe and the labeled macromolecule. An increase in size of the labeled macromolecule through binding to another macromolecule can be measured directly by fluorescence anisotropy. The observed value of anisotropy is weighted in accordance to the fraction of free and bound labeled macromolecule. Thus, fluorescence anisotropy is a versatile technique to monitor macromolecular assembly and had been employed to monitor DNA-protein interaction (3032), assembly of the DNA replication machinery (33), dynamics of domain-domain interactions (34, 35), and protein-protein interaction (36). This approach has been developed recently as a high throughput assay for G protein-coupled receptor binding (37).
Mass Spectrometry
New developments in mass spectrometry are particularly useful in studying and quantifying the dynamics of macromolecular assembly. The approach of nanoflow electrospray coupled with time-of-flight mass spectrometry (38) allows one to study macromolecular assembly in its native state, and the range of molecular weights of the complex has been extended to 106 Da, e.g. Escherichia coli ribosome (39). Not only can the mass of the final product of the assembly process be determined, but the approach has been applied successfully to define the macromolecular organization of the Yersinia peatis capsular F1 antigen (40), the chaperone complex from Methanobactrium thermoautotrophicum (41), the post-synaptic density-95 complex (42), and the yeast nuclear pore complex (43).
The versatility of the approach enables one to define the noncovalent pathways of assembly and disassembly of these macromolecular assemblies (44, 45). In the study of E. coli ribosomes, Robinson and co-workers (45) were able to detect the dissociation of some ribosomal proteins from the complex. Although at this stage of development thermodynamic parameters have not been derived from these observations, the mass spectrometric observation correlates qualitatively with the known reactions of these species (45). In other studies using electrospray ionization mass spectrometry, conditions have been worked out to determine thermodynamic parameters, i.e. the Kd of the interaction. The Kd values of the E. coli replication-inhibiting protein Tus and its mutants for the specific Ter DNA sequence in the nM to µM concentration range were determined (46) in agreement with classical solution studies. Thus, these developments are most encouraging to investigators who are interested in monitoring the pathways of signal transmission between macromolecules and the perturbation of the pathways as a consequence of further interaction with ligands or other biomacromolecules.
Other Methods and Combinations of Approaches for Protein Characterization
New technology, such as biosensors based on microfluiditics and surface plasmon resonance (47), and revitalized methods, such as computerized isothermal titration microcalorimetry (48), also provide rigorous and powerful methods tk;2for determining dissociation constants and/or kinetics of macromolecular interactions. A recent paper (47) has documented that judicious use of the biosensor to measure binding on a surface can provide data equivalent to, or better than, solution methods of analysis. Robotics has maximized throughput of the Biacore biosensor and could potentially do the same for microcalorimetry.
A combination of these methods is frequently the most effective means to characterize proteins. Dynamic light scattering studies, along with gel filtration chromatography, analytical ultracentrifugation, and chemical cross-linking experiments, support the model that in vivo copper loading of yeast superoxide dismutase SOD1 occurs via a heterodimeric intermediate with its copper chaperone, rather than between two homodimers (49). Stopped-flow, static, and dynamic light scattering studies of vesicular stomatitis virus nucleocapsid proteins were used to determine their conformation, extent of self-association, and amount of bound matrix protein from the radius of gyration, concentration dependence of the apparent molecular mass, and diffusion coefficient (29). Dynamic light scattering, sedimentation, equilibrium, and circular dichroism measurements provided evidence that the anti-apoptotic protein BAG-1 exists as an elongated, highly helical monomer in solution (50). Isothermal titration microcalorimetry confirmed the 1 to 1 stoichiometry of the heterodimer with Hsp70, and both microcalorimetry and surface plasmon resonance yielded a Kd of 100 nM for the complex that modulates chaperone activity (50).
Receptors and Receptor Extracellular Domains
Dimerization is viewed frequently as either a prerequisite or a required step in receptor activation, and receptors frequently bind several other proteins upon activation. The proteomic approach has been successful in identifying bound cellular proteins, e.g. 77 proteins have been identified as binding to the N-methyl-D-aspartate receptor complex (51). However, study of many individual receptor systems has focused on quantitative analysis of the association of the receptor extracellular domain (ECD), either with or without its ligand, that frequently provides the driving force for receptor dimerization. These latter studies demonstrate the types of quantitative information required for understanding how hormones and growth factors trigger cell signaling. Demonstration of an interaction or the determination of the three-dimensional structure by crystallography or NMR, although an extremely useful initial step, does not provide the entire picture without a more quantitative assessment.
Nerve growth factor receptor (NGF) and the neurotrophin family that includes NGF, brain-derived neurotrophic factor, neurotrophin 3, and neurotrophin 4 initiate cellular responses by binding to a series of cognate protein tyrosine kinase receptors TrkA, TrkB, and TrkC (52). Several studies have examined the interaction of the dimeric neurotrophin with the recombinant full-length ECD of the cognate receptor. Evidence has been provided from sedimentation equilibrium analysis and size exclusion chromatography that the full ECD of TrkB and TrkC form a complex with BDNF and NT3, respectively, with a stoichiometry of either 2:1 or 1:1 (receptor to neurotrophin dimer) depending on concentration (53). However, the dissociation constants determined were about three orders of magnitude higher than that expected from bioassay and cellular binding studies. Also, size exclusion chromatography, in conjunction with light scattering, has indicated that the TrkA ECD is monomeric and dimerizes in the presence of NGF (54). This interaction coincided with a small but significant conformational change as determined by CD studies. Although binding of NGF to TrkA-ECD was measured with a surface plasmon resonance biosensor, no direct determination of the Ka for receptor dimerization was measured in these latter studies.
In contrast, crystallographic studies have shown that the d5 IgG-like subdomain of TrkA forms an incorrect dimer in which symmetric domain swapping has occurred such that one ß-strand of one TrkA chain has folded out to form a strand with the same ß-sheet in the accompanying subunit (55). Such an interaction would not be able to bind NGF and would thus provide incorrect binding constants when used for solution studies. Whether such a false dimer will occur in the full-length ECD, which includes two cysteine subdomains, a leucine-rich subdomain, and two IgG-like subdomains, is unknown. Additional studies in solution of full-length ECDs of the Trk receptors should provide valuable information in this regard and correlate the structural and the biophysical data.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Published, MCP Papers in Press, June 24, 2002, DOI 10.1074/mcp.R200003-MCP200
1 The abbreviations used are: CD, circular dichroism; ECD, extracellular domain; NGF, nerve growth factor.
* This work was supported in part by National Institutes of Health Grants NS24380 (to K. E. N.), NS36700 (to K. E. N.), and GM-45579 (to J. C. L.) and by Robert A. Welch Foundation Grants E-013 and E-1238 (to J. C. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Finch UHS/Chicago Medical School, 3333 Green Bay Rd., N. Chicago, IL 60064. Email: neetk{at}finchcms.edu
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REFERENCES |
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