The Importance of Additive and Non-Additive Mutational Effects in Protein Engineering

摘要

Following the development of site-specific mutagenesis by Michael Smith more than three decades ago, scientists have at their disposal a molecular biological method with which a given amino acid at a defined position of a protein can be substituted by any one of the other 19 canonical representatives, This technique has proven to be invaluable in the study of enzyme mechanisms. The enormous progress made in attempting to understand the intricacies of enzyme mechanisms by using this technique coupled with an arsenal of other experimental and computational methods developed during the last few decades has shown that the "secrets" behind these biocatalysts are more complex than originally thought. In addition to clearing up the gross features of a given enzyme mechanism, phenomena such as protein dynamics and flexibility, electrostatic preorganization, tunneling, and allosteric effects need to be considered and evaluated case by case.141 This makes the detailed interpretation and prediction of mutational effects difficult and, as highlighted in this Essay, especially challenging if several point mutations are introduced.