Determinants of protein stability and function inferred from saturation mutagenesis and next-generation sequencing

Abstract

Proteins play a central role in all biological processes. Despite a similar underlying composition, they exhibit immense diversity in structures and functions, a subject of great interest. The research presented in this thesis aims to deepen our understanding of the relationships between protein structure and function by describing various techniques developed to investigate these relationships. Deep sequencing of synonymous mutations in the E. coli CcdB toxin revealed complex interactions affecting protein expression and function, highlighting the nuanced roles of translational control and codon specificity. RNA sequencing provided insights into the differential gene expression and RNA cleavage specificities of various toxins in M. tuberculosis, enhancing our understanding of the physiological roles of these toxin-antitoxin systems. Our studies on the MazEF6, MazEF9, VapBC13, and VapBC26 systems have provided valuable insights into their regulatory roles and RNA cleavage patterns.