Site-selective mutagenesis

Protein engineering is now routinely used to modify protein molecules either via site-directed mutagenesis or by combinatorial methods. Factors that are Important for the stability of proteins have been studied, such as stabilization of a helices and reducing the number of conformations in the unfolded state. Combinatorial methods produce a large number of random mutants from which those with the desired properties are selected in vitro using phage display. Specific enzyme inhibitors, increased enzymatic activity and agonists of receptor molecules are examples of successful use of this method. 

In molecular pharmacology research an indirect proof of a structural model is possible by functional examinations, e.g., by molecular biological experiments. Well-selected site directed mutagenesis and their functional characterization allows confirmation or rejection of a molecular protein model. The process is organized as an iterative procedure, where the biological answer of suggested mutations is used to refine the model. The iteration continues until the model... 

Fig. 2.5. Possible applications of a coupling parameter, A, in free energy calculations, (a) and (b) correspond, respectively, to simple and coupled modifications of torsional degrees of freedom, involved in the study of conformational equilibria (c) represents an intramolecular, end-to-end reaction coordinate that may be used, for instance, to model the folding of a short peptide (d) symbolizes the alteration of selected nonbonded interactions to estimate relative free energies, in the spirit of site-directed mutagenesis experiments (e) is a simple distance separating chemical species that can be employed in potential of mean force (PMF) calculations and (f) corresponds to the annihilation of selected nonbonded interactions for the estimation of e.g., free energies of solvation. In the examples (a), (b), and (e), the coupling parameter, A, is not independent of the Cartesian coordinates, x. Appropriate metric tensor correction should be considered through a relevant transformation into generalized coordinates...

The selective monohydroxylation of heterocyclic compounds such as piperidine derivatives1741 and the 7-lactam (19)[751 have been studied. It is also been shown that hydroxylation of phenylcyclohexane can be effected using cytochrome P450 and the regioselectivity of hydroxylation can be altered by site-directed mutagenesis of the enzyme1761. 

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