Deep Mutational Scanning Analyses of Protein Stability and Function

Abstract

Protein stability is critical to understanding protein function, folding, and interactions within living organisms. It plays a key role in protein engineering, where designing stable proteins is essential for developing biopharmaceuticals like antibodies and vaccines. This thesis investigates various methodologies to assess protein stability, emphasizing yeast surface display (YSD) and deep sequencing approaches. The study begins by exploring traditional in vitro, in lysate, and in silico stability assessment techniques, highlighting each method's strengths and limitations. Recent advances in high-throughput sequencing and deep mutational scanning have revolutionized the field, enabling comprehensive analysis of protein variants and identification of stabilizing mutations on a large scale. The focus of the research is on advantage of using YSD method to determine the thermal stability of proteins. The study compares the thermal stability of mutants of the bacterial toxin CcdB and the Receptor Binding Domain (RBD) of the SARS-CoV-2 spike protein, using both YSD and differential scanning fluorimetry. The strong correlation between the results obtained from these methods validates YSD as an effective tool for determining protein stability without the need for purification. For proteins that exhibit reversible thermal denaturation, such as GB1, the research employs an alternative approach using chemical denaturation with guanidine hydrochloride to prevent refolding. This method provides stability values comparable to those obtained through conventional techniques. Additionally, the thesis introduces a novel approach for identifying stabilizing mutations by externally perturbing the YSD library with chemical denaturants. This strategy successfully identifies stabilizing mutations in GB1 by analyzing binding intensities using fluorescence-activated cell sorting (FACS) and deep sequencing. Further investigation extends to specific buried site mutants of the ccdB gene, where co-expression with the interacting partner CcdA within the ccdAB operon alleviates folding defects. The study suggests that the CcdAB complex assembles cotranslationally, providing a mechanism to counteract deleterious mutations. Finally, the thesis explores the development of a polyantitoxin strategy to simultaneously neutralize multiple Type II Toxin-Antitoxin (TA) modules. This approach, tested in E. coli and extended to M. tuberculosis, demonstrates the potential of polyantitoxins as tools for modulating multiple TA systems, offering valuable insights into their roles in bacterial persistence and drug tolerance. Overall, this research enhances the understanding of protein stability and its applications, particularly in protein engineering and the study of bacterial TA systems.