Protein engineering folding

Finkelslain A V 1997. Can Protein Unfolding Simulate Protein Folding Protein Engineering 10 843... 

Orengo C A, T P Flores W R Taylor and J M Thornton 1993. Identification and Oassificalion of Prc Fold Families. Protein Engineering 6 485-500. 

Noncovalent Forces Stabilizing Protein Structure. Much of protein engineering concerns attempts to alter the stmcture or function of a protein in a predefined way. An understanding of the underlying physicochemical forces that participate in protein folding and stmctural stabilization is thus important. 

Through combined effects of noncovalent forces, proteins fold into secondary stmctures, and hence a tertiary stmcture that defines the native state or conformation of a protein. The native state is then that three-dimensional arrangement of the polypeptide chain and amino acid side chains that best facihtates the biological activity of a protein, at the same time providing stmctural stabiUty. Through protein engineering subde adjustments in the stmcture of the protein can be made that can dramatically alter its function or stabiUty. 

Recently Alan Fersht, Cambridge University, has developed a protein engineering procedure for such studies. The technique is based on investigation of the effects on the energetics of folding of single-site mutations in a protein of known structure. For example, if minimal mutations such as Ala to Gly in the solvent-exposed face of an a helix, destabilize both an intermediate state and the native state, as well as the transition state between them, it is likely that the helix is already fully formed in the intermediate state. If on the other hand the mutations destabilize the native state but do not affect the energy of the intermediate or transition states at all, it is likely that the helix is not formed until after the transition state. 

What can be done by predictive methods if the sequence search fails to reveal any homology with a protein of known tertiary structure Is it possible to model a tertiary structure from the amino acid sequence alone There are no methods available today to do this and obtain a model detailed enough to be of any use, for example, in drug design and protein engineering. This is, however, a very active area of research and quite promising results are being obtained in some cases it is possible to predict correctly the type of protein, a, p, or a/p, and even to derive approximations to the correct fold. 

The ultimate goal of protein engineering is to design an amino acid sequence that will fold into a protein with a predetermined structure and function. Paradoxically, this goal may be easier to achieve than its inverse, the solution of the folding problem. It seems to be simpler to start with a three-dimensional structure and find one of the numerous amino acid sequences that will fold into that structure than to start from an amino acid sequence and predict its three-dimensional structure. We will illustrate this by the design of a stable zinc finger domain that does not require stabilization by zinc. 

Fersht, A. R., Matouschek, A., and Serrano, L. (1992). The folding of an enzyme. 1. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771-782. 

Ollis, D.L., Cheah, E., Cygler, M. etal. (1970) The alpha/beta hydrolase fold. Protein Engineering, 5, 197-211. 

Transient folding intermediates characterized by protein engineering. Nature, 346, 440-445. 

The fastest-folding small proteins generally fold on much slower time scales than the time scale of formation of secondary structure. The speed record is currently held by lambda(6-85), a truncated, monomeric form of the N-terminal domain of lambda repressor, which refolds with a half-life of approximately 140 fjis. A thermostable lambda(6-85) variant with alanine substituted for glycine residues 46 and 48 in the third helix folds faster in dilute solutions of de-naturant, with an extrapolated half-life of less than 10 /us in water.13 Cold-shock protein CspB from Bacillus subtilis folds in about 1 ms.61 Engineered mutants of the P22 Arc repressor62 and CI263 fold in a fraction of a millisecond. 

There are now significant advances toward this goal. Experimentalists are able to alter the activity and stability of proteins by protein engineering, and the first tentative steps in protein design are under way. Theoreticians are able to simulate many aspects of folding and catalysis with increasing detail and reliability. 

There is a severe practical problem in looking for correlations between the rate constants for folding of small proteins and their structural or thermodynamic properties—specific structural features can dominate the rate of folding. For example, we know from the protein engineering studies on barnase and CI2 that specific mutations can slow down the rate of folding by several orders of... 

Phillips, D C. Protein Engineering/ Review (Umv. of Wales), 46 (March 1987). Richards, F.M. The Protein Folding Problem, ScL Amer., 54 (January 1991). Radousky, H.B., G. Hammond, Z. Xu, et al. Gene Families Studies of DNA, RNA, Enzymes and Proteins, World Scientific Publishing Company. Inc., River Edge, NJ, 2001. 

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