Experimental Protein Folding

Investigation of the equilibrium between native and unfolded conformations formed on solvent denaturation, temperature, and pressure have now been performed on a wide range of proteins. For example, James and Sawan have studied the effect of increasing guanidinium chloride concentration on the mobility of individual histidine residues in ribonuclease (pancreatic) C n.m.r. spectroscopy using spin-lattice relaxation in an off-resonance rotating frame. They found that L-histidines-12, -105, and -119 increase in mobility up to a denaturant concentra- 

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C. D. Snow, H. Nguyen, V. S. Pande, and M. Gruebele. Absolute comparison of simulated and experimental protein folding dynamics. Nature, 420 102-106, 2002. 

Experimental protein folding data based on both x-ray crystallographic and NMR results are available online through the Protein Data Bank (PDB, http //www.rcsb.org/pdb). 

Predictions from these model studies match well with results on experimental protein folding studies of some real proteins (Ortiz and Skolnick, unpublished). Indeed, when a similar procedure is employed on real proteins for which experimental data are available, there is a substantial overlap between the folding nucleus found experimentally and the folding nucleus predicted from the multivariate analysis of multiple sequence alignments. 

Finding the minimum of the hybrid energy function is very complex. Similar to the protein folding problem, the number of degrees of freedom is far too large to allow a complete systematic search in all variables. Systematic search methods need to reduce the problem to a few degrees of freedom (see, e.g.. Ref. 30). Conformations of the molecule that satisfy the experimental bounds are therefore usually calculated with metric matrix distance geometry methods followed by optimization or by optimization methods alone. 

When we have the information from the sequencing of the human genome, and want to understand the properties of those proteins that are coded by some of the genes but not yet known experimentally, we need to solve the protein-folding problem. Then we can translate the gene sequence—which specifies the sequence of amino acids—into the three-dimensional structure of the unknown protein. 

Karplus M. and Weaver D. L. Protein-folding dynamics - the diffusion-collision model and experimental-data. Prot. Sri. (1994) 3(4) 650-668. 

Folding to native-like structure has been demonstrated with fragments of jS-galactosidase, lysozyme, serum albumin, penicillinase, and tryptophan synthetase. The capability of protein fragments for independent formation of structure therefore has substantial experimental basis. This generalization also makes plausible the idea that, in general, protein folding occurs by parts, that is, in a modular fashion. 

In the final analysis protein folding will be really understood only with the aid of much more extensive, direct experimental evidence. Speculative hypotheses can be useful, however, in suggesting potentially fruitful questions for experimental investigation. Probably the most important idea suggested by the above schema is that there are likely to be considerable systematic differences in the kinetics of folding between the various major structural categories of proteins. 

In addition to identifying protein partners, yeast two-hybrid technology can be used to identify and study in detail the interaction domains between two proteins. Here, bait and/or fish truncation or deletion constructs of the parent proteins are engineered and characterized as described earlier (see 3.1 Selection and characterization of bait constructs). These are then investigated for association in a yeast two-hybrid interaction assay. Once the BD has been identified, it can be further refined by mutagenesis. The same caveat applies to these studies as for the identification of associating proteins, i.e., it is assumed that the respective fusion proteins fold and adopt the same or a similar three-dimensional conformation to the native protein. This is not always the case and results should be interpreted with caution and if possible, always validated by an alternative experimental approach. O Table 19-1 shows an example of mapping the... 

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