Mechanistic insights into protein stabilization and protein protein interactions

Abstract

Most amino acid substitutions in a protein either lead to partial loss of function or are near neutral. Several studies have shown the existence of second-site mutations that can rescue the defects caused by diverse loss of function mutations. Such global suppressor mutations are key drivers of protein evolution. However, the mechanisms responsible for such suppression remain poorly understood. In the present work, we used CcdB (Controller of Cell Death or Division), a protein from E.coli, as the model protein to understand the precise mechanisms of action of these compensatory global suppressor mutations. Chapter 1 briefly outlines the nature of protein sequence structure relationships and the use of several computational and experimental approaches to estimate protein stability and identify protein-protein interaction sites. The conventional methodologies available to measure protein stability and kinetics require large amounts of protein and are time consuming. In Chapter 2, we provide a comparative study of the use of a nano Differential Scanning Fluorimeter (nanoDSF) with that of the conventional fluorimeter (Chattopadhyay and Varadarajan, 2019) to measure stability and folding kinetics. Chapter 3 provides detailed mechanistic insight into the rescue of stability and functional defects caused by inactive mutations present in the core of the protein by global suppressor substitutions, using the bacterial toxin CcdB as the model protein. Chapter 4 describes the extension of the study of global suppressor substitutions in a number of other protein systems which belong to varied classes and have a wide range of functionalities, We observed that the stabilization caused by global suppressor substitutions in the background of the folding defective, loss of function mutants was significant, and was primarily achieved through an increase in the kinetic parameters of refolding. These observations hold true across diverse protein systems, suggesting the results are general. Chapter 5 describes a high throughput metho...