Exploring the folding and dynamics of nascent proteins

Previous in vitro protein-folding studies produced invaluable results. However, current medical questions press for an understanding of protein folding in vivo, from birth to death in a protein's lifecycle. This thesis work addresses the dynamics of ribosome-bound nascent polypeptides as a function of elongation stage, local environment and interaction with chaperones by time-resolved fluorescence anisotropy monitored via frequency-domain fluorometry.;Chapter 1 provides a brief overview of the protein folding field and the spectroscopic approaches pertinent to this study.;Chapter 2 addresses the dynamics of the single-domain alpha-helical protein apomyoglobin at different stages of its chain elongation on the ribosome, with emphasis on the timescales of each of the detectable motions. In addition to extremely slow (mus) and fast (sub-ns) motions, a peculiar independent local dynamics, occurring on the 1-7 ns timescale, is detected for intermediate and long nascent protein fragments. Such type of motion is sequence-specific.;Chapter 3 focuses on the spatial confinement of the local motions experienced by the nascent natively unfolded protein PIR. Stochastic dynamics across a cone (according to a square-well potential) is assumed. We show that the ribosome constrains the amplitude of nascent PIR's dynamics both inside and outside the ribosomal tunnel.;Chapter 4 addresses the spatial confinement of the local motions of nascent apomyoglobin as a function of the presence/absence of cotranslationally active chaperones and the ribosome. This chapter also expands the mathematical model described in Chapter 3 to include the presence of a second local motion. One of the major findings of this work is that the small amplitude of the sub-ns motions is not imposed by the chaperones but by the very nature of the nascent chain and the ribosomal surface.;Chapter 5 compares different methods to induce the release of nascent polypeptides from the ribosome. Among such methods, is a novel mild approach involving treatment with hydroxylamine. This reagent acts by performing a nucleophilic attack on the ester bond linking the tRNA to the nascent polypeptide.;Chapter 6 provides a brief survey of plausible future directions of the research described in this thesis work.