Electrostatic Effect of the Ribosomal Surface on Nascent Polypeptide Dynamics

Abstract

The crucial molecular events accompanying protein folding in the cell are still largely unexplored. As nascent polypeptides emerge from the ribosomal exit tunnel, they come in close proximity with the highly negatively charged ribosomal surface. How is the nascent polypeptide influenced by the ribosomal surface? We address this question via the intrinsically disordered protein PIR and a number of its variably charged mutants. Two different populations are identified: one is highly spatially biased, and the other is highly dynamic. The more negatively charged nascent polypeptides emerging from the ribosome are richer in the extremely dynamic population. Hence, nascent proteins with a net negative charge are less likely to interact with the ribosome. Surprisingly, the amplitude of the local motions of the highly dynamic population is much wider than that of disordered polypeptides under physiological conditions, implying that proximity to the ribosomal surface enhances the molecular flexibility of a subpopulation of the nascent protein, much like a denaturing agent would. This effect could be important for a proper structural channeling of the nascent protein and the prevention of cotranslational kinetic trapping. Interestingly, a significant population of the highly spatially biased nascent chain, probably interacting extensively with the ribosome, is present even for very negatively charged nascent proteins. This "sticking" effect likely serves to protect nascent proteins (e.g., from cotranslational aggregation). In all, our results highlight the influence of the ribosome in nascent protein dynamics and show that the ribosome's function in protein biogenesis extends well beyond catalysis of peptide bond formation.

Nonorthogonal tRNAcysAmber for protein and nascent chain labeling

“…Fluorescence spectroscopy of ribosome-bound nascent chains (RNCs) has recently been used to increase our understanding of aspects of protein biogenesis, such as protein trafficking (Flanagan et al 2003;Woolhead et al 2004;Holtkamp et al 2012), chaperone recognition (Lin et al 2012), and cotranslational protein folding (Ellis et al 2008;Knight et al 2013). Unfortunately, the presence of the ribosome hampers standard labeling of RNCs, necessitating the use of alternative strategies.…”

“…In the case of the former, site-specific incorporation of a single fluorophore is largely restricted to rare proteins with a single lysine (Lin et al 2012), because lysine residues are highly abundant in proteins. The use of tRNA fMet is also limited, as it allows only N-terminal protein labeling (Ellis et al 2008;Knight et al 2013). …”

Entropy Drives the Formation of Salt Bridges in the Protein GB3

“…[1] They play an important role in the binding of proteins to small ligands, [2] proteins, [3] DNA, [4] and other molecules. [5] It has also been shown that protein thermostability can be improved through optimizing the surface charge-charge interactions. [6] As two ionizable residues with opposite charges approach to each other,as alt bridge is formed.…”

The ribosome regulates flavodoxin folding

“…As the exterior of the ribosome consists for a large part of rRNA, it is negatively charged in many areas. Negative charge also surrounds the exit tunnel, thereby providing an environment that is very different from the cytoplasm (177). This charge may influence protein folding, as it attracts positively charged residues of the nascent chain and repels negatively charged ones (170).…”

“…This charge may influence protein folding, as it attracts positively charged residues of the nascent chain and repels negatively charged ones (170). It also restricts the dynamics of nascent chains according to their charge (177). Due to these electrostatic interactions, nascent chains may be protected from misfolding or aggregation.…”