Calculation of Mutagenesis Hot-Spots

In focused mutagenesis experiments, the challenge is to identify the residues where mutagenesis is likely to be beneficial. Indeed, many successful directed evolution experiments show that mutations occur in regions that would be hard or impossible to predict (and difficull to explain that they do), even when a high-resolution structure and much information about the enzyme is available 14, 98-1001. One possibility is to make use of knowledge gained from early rounds of random point 

Alanine scanning has been widely used to identify the residues which are contributing to various protein properties[105, 10S1. Alanine substitutions are made at various positions and the perturbation in the property of interest is measured. This has several potential applications to directed evolution. For instance, it can be used to predetermine which positions are essential to the structure (or function) of the protein and therefore should be avoided. Conversely, positions that tolerate the alanine substitutions may be good candidates for saturation mutagenesis. Unfortunately, this procedure is tedious. To surmount this difficulty, Kollman and coworkers proposed a method to determine the effects of alanine substitutions computationally11071. Kollman s method could be used to scan the protein structure for positions to mutagenize in directed evolution. 

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