Applying advanced computational and experimental tools to explore surface electrostatics in chitinases

Understanding how proteins work is a major challenge in the post-genome era. Traditionally proteins have been studied by the use of numerous biochemical techniques, but as the number of proteins that needs to be characterized has increased, so has the num ber of in computo techniques to study protein structure and function. Many techniques for modeling and analyzing protein structures exist, but in computo analysis of several crucial properties of proteins still remains a major challenge. In this project w e will use protein electrostatics calculations and molecular dynamics simulations to address important aspects of protein structure and function. We will do this by an integrated experimental and computational approach, using a well-studied chitinase from Serratia marcescens (Chitinase B, ChiB) as a model protein. Previous work has shown that catalysis in this enzyme depends on a complex network of (longer-range) electrostatic interactions, which makes ChiB is an interesting target for further exploration of the importance of protein electrostatics. Furthermore, the catalytic domain of ChiB has a (beta/alpha)8 fold (also known as a TIM barrel fold) which is one of the most common folds in enzymes. The experimental work will consist of semi random site-di rected mutagenesis of the ChiB surface, combined with gene shuffling and high-throughput screening of mutants with relevant changes (primarily pH-activity and pH-stability profiles, cold activity, and temperature stability). The computational work will fo cus on calculations of pKa values and electrical fields, as well as on molecular dynamics simulations. We will also attempt to perform computational analyses of the relationship between dynamics and electrostatics. The computational work will be done in c ollaboration with one national and a foreign expert laboratory.