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 & 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 methodology called Ter-Seq to identify mutations that likely stabilize transient ternary complexes CcdA-CcdB-Gyrase was the model system and mutational effects were probed using yeast surface display coupled to FACS and Sanger sequencing. The method was also used to isolate CcdB mutants that are either resistant or more sensitive to CcdA mediated rejuvenation than WT CcdB. Chapter 6 describes the biophysical characterisation of several type II toxin:antitoxin systems of Mycobacterium tuberculosis, namely MazEF3, MazEF6, MazEF9, VapBC3, VapBC4 and VapBC21, and explores possible cross talk between non-cognate toxin-antitoxin pairs. Chapter 7 describes a rapid method of mapping interfacial residues in the MazEF3, MazEF6 and MazEF9 protein complexes using cysteine scanning mutagenesis coupled to FACS and deep sequencing.