Protein Engineering mutations

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. 

Kunichika, K., Hashimoto, Y. and Imoto, T. (2002) Robustness of hen lysozyme monitored by random mutations. Protein Engineering, 15, 805-809. 

Hamamatsu, N., Aita, T., Nomiya, Y. et al. (2005) Biased mutation-assembling an efficient method for rapid directed evolution through simultaneous mutation accumulation. Protein Engineering Design Selection, 18, 265-271. 

Finally, a strategy to exploit protein engineering to mutate the ATP-binding pockets of protein kinases with the objective of enhancing selectivity for synthetic ATP analogs or inhibitors has been developed [66-68] using Src tyrosine kinase as a prototype model. In brief, mutation of a conserved amino acid in the ATP binding pocket was made to create a unique new site... 

Teplyakov, A. V., van der Laan, J. M., Lammers, A. A., Kelders, H., Kalk, K. H., Misset, O., Mulleners, L.J. Dijkstra, B. W. (1992). Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus functional and structural consequences of mutation at the S4 substrate binding pocket. Protein Engineering, 5, 413-20. 

It is difficult to attribute quantitatively by experiment the rate enhancements of the different factors contributing to catalysis. Protein engineering can get close to accurate answers when dealing with nonpolar interactions, especially in subsites. But analysis of mutation is at its weakest when altering residues that interact with charges (Chapter 15). The next development must be in improved methods of computer simulation. Controversies arise when there are no intermediates in the reaction because the kinetics can fit more than one mechanism. Again, computer simulation will provide the ultimate answers. 

A model was proposed for the structure of a productively bound enzyme-substrate complex, extrapolated from the structures of inert complexes (Figure 16.6). Tyr-248 had been implicated in catalysis from early chemical modification experiments. But one of the first protein engineering experiments, the mutation Tyr-248 —> Phe, showed that it is not involved.149... 

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... 

Even if we restrict our design to a small number of sites in the protein, the combinatorial possibilities quickly approach astronomical dimensions. If we consider mutations at 10 sites and a subset of 10 amino acids, we have 1010 possible variants. Although experimental approaches are under development that can actually search large subsets of protein sequence space, it is not at all a small feat to identify those variants that give rise to a stable structure and at the same time come close to the desired features. Therefore, computational approaches that, with some reliability, are able to pick those variants having a stable structure are desirable instruments in the protein engineer s toolbox. 

The design of supramolecular catalysts may make use of biological materials and processes for tailoring appropriate recognition sites and achieving high rates and selectivities of reactions. Modified enzymes obtained by chemical mutation [5.70] or by protein engineering [5.71] represent biochemical approaches to artificial catalysts. 

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