Understanding structure, function, and dynamics in macromolecular complexes

Abstract

In the scope of my thesis work, a concerted effort was undertaken to elucidate bi- ological functions through the lens of structural analysis, employing protein dy- namics. The role of protein dynamics in shaping and facilitating protein function, by enabling exploration of the conformational landscape, was the primary objec- tive. Proteins seldom function in isolation and they often engage in complexes with other macromolecules such as other proteins, DNA, RNA, and small molecules. In this chapter, I also give a comprehensive review of diverse computational tools employed in the scrutiny of protein-macromolecule interactions. Recent progress in computational resources has expanded the number of tools available for study- ing protein-macromolecule interactions at various molecular levels. These include tools for predicting interacting residues from primary sequences, modeling protein- macromolecule complexes, predicting hotspots in these complexes, and insights into understanding the dynamics of their interactions. Here I present an overview of var- ious computational tools to study different aspects of protein-protein and protein- RNA interactions, with a focus on the overall application and development of the field. The second chapter of the thesis was dedicated to the exploration of inter-protein bifurcated interactions. The study addressed the less-explored domain of interfa- cial residues, i.e., inter-protein bifurcated interactions. While interfacial residues are known to confer stability and specificity to these interactions, the extent of their participation in inter-protein bifurcated interactions and the contribution of these interactions is not well understood. This study highlighted the importance of these interactions in multi-protein complexes. Subsequently, the third chapter delved into the molecular intricacies of bacte- rial two-component systems. Comprising sensor kinase and response regulator pro- teins, these systems orchestrate bacterial responses to environmental fluctuations. Predominantly characterized by cognate pairs, an intriguing phenomenon of non- cognate interactions was observed in M. tuberculosis. This chapter is focused on the structural basis and specificity of this atypical crosstalk. The fourth chapter embarked on an exploration of the dynamic alterations within the RNA polymerase complex upon interaction with RbpA. In the context of M. tu- berculosis, the RNA polymerase (RNAP) complex encompasses subunits 2α, β, β’, and ω. This complex further interacts with transcription activators and factors like RbpA and CarD. This study was centered on unraveling the structural and dynamic changes within the RNAP complex as it interacts with RbpA, DNA, and CarD. The insights shed light on the intricate interplay among transcription factors during tran- scription initiation, thus offering a lens into the regulatory mechanisms. The fifth chapter ventured into the intricacies of the impact of mRNA on the 30S initiation complex and its preparatory role for interaction with the incoming xvi 50S subunit. Recognizing the regulatory role of mRNA in protein synthesis kinetics, the research dissected the structural, mechanistic, and dynamic nuances of mRNA recognition within the ribosome. This was accomplished through a comparative analysis of ribosomal complexes in the presence and absence of mRNA. This study highlighted the significance of mRNA binding in the ribosome complex and putative allosteric sites I identified in regions away from mRNA binding sites in the complex. This work provides fresh insights into mRNA association with the ribosome, high- lighting changes in the interactions and dynamics of the ribosome assembly because of the binding. In the concluding chapter, an overall view of all the projects was provided. The research spotlight was aimed at comprehending the intricate interplay of dynamics within protein-macromolecule assemblies, particularly in the context of large com- plexes. The thesis traversed diverse regions of computational analysis to elucidate the roles played by various protein complexes in cellular functions. Through the systematic exploration of protein dynamics, inter-protein interactions, bacterial two- component systems, RNA polymerase dynamics, protein-RNA interactions, and ri- bosomal responses to mRNA binding, the study deepened the understanding of fundamental biological processes.