Insights into some key aspects of protein folding I. Relation between protein folding and aggregation under physiologically relevant conditions II. Probing the influence of newly synthesized proteins on ribosomal components
We still know very little about protein folding in the cell and what parameters and molecular machines cause soluble proteins to achieve their native conformation. Although Anfinsen’s thermodynamic hypothesis explains the origin of protein stability, it does not account for the presence of protein aggregates and where they lie in an energy landscape relative to the native state, under physiologically relevant conditions. Protein folding starts within the ribosome during biosynthesis. While the ribosome and cotranslationally active molecular chaperones help nascent chains achieve their native state, little is known about the influence of nascent polypeptides and proteins on the ribosome and its apparent stability. In this thesis I show that Anfinsen’s thermodynamic hypothesis needs to be modified to include soluble and insoluble protein aggregates because most proteins are kinetically trapped relative to their aggregated states. In addition, I explore some of the determinants of the apparent stability of the 70S ribosome in E. coli. This thesis is divided into four chapters. Chapter 1 explains Anfinsen’s thermodynamic hypothesis together with its major implications, and outlines the major aspects of the ribosome structure and function. This chapter serves as the conceptual background for the remaining portions of this document. Chapters 2 and 3 explore the concept of protein kinetic trapping under physiologically relevant conditions and demonstrates the need to extend Anfinsen’s hypothesis to include protein aggregates. More specifically, Chapter 2 introduces an experimental strategy denoted as the “cyclic perturbation approach” to show that two structurally distinct, non-amyloid aggregate states of sperm whale apomyoglobin are kinetically trapped relative to each other and relative to the native state. The experiments presented in Chapter 3 show that kinetic trapping is a more general phenomenon than previously thought, and that it can be extended to the large majority of soluble proteins in E. coli. A series of kinetic simulations confirms this phenomenon. Chapter 4 showcases the influence of the nascent chain on the apparent stability of the 70S E. coli ribosome. The presence of the newly synthesized polypeptide was found to stabilize the ribosome. Surprisingly, characteristic properties of the nascent chain, including its N- and C-terminal residues, net charge, and length (beyond 32 residues) do not have any influence on the structural stabilization of the 70S complex by the nascent polypeptide. This chapter also introduces a working model for denaturant-induced ribosomal disassembly. Overall, this dissertation shows that the kinetic trapping of native, unfolded and intermediate protein states relative to aggregates under physiologically relevant conditions is a general phenomenon in Nature. In addition, it shows that nascent proteins contribute to increasing the apparent stability of the bacterial ribosome.