Structure and dynamics of a ribosome-bound nascent chain by NMR spectroscopy

Abstract

Protein folding in living cells is inherently coupled to protein synthesis and chain elongation. There is considerable evidence that some nascent chains fold into their native structures in a cotranslational manner before release from the ribosome, but, despite its importance, a detailed description of such a process at the atomic level remains elusive. We show here at a residue-specific level that a nascent protein chain can reach its native tertiary structure on the ribosome. By generating translation-arrested ribosomes in which the newly synthesized polypeptide chain is selectively 13 C/ 15 Nlabeled, we observe, using ultrafast NMR techniques, a large number of resonances of a ribosome-bound nascent chain complex corresponding to a pair of C-terminally truncated immunoglobulin (Ig) domains. Analysis of these spectra reveals that the nascent chain adopts a structure in which a native-like N-terminal Ig domain is tethered to the ribosome by a largely unfolded and highly flexible C-terminal domain. Selective broadening of resonances for a group of residues that are colocalized in the structure demonstrates that there are specific but transient interactions between the ribosome and the N-terminal region of the folded Ig domain. These findings represent a step toward a detailed structural understanding of the cellular processes of cotranslational folding.cotranslational folding

Kinetic modelling indicates that fast-translating codons can coordinate cotranslational protein folding by avoiding misfolded intermediates

“…A key challenge in this context is to interpret and predict the influence of individual codon translation rates on cotranslational protein folding and misfolding. The benefits of responding to this challenge are manifold: it would ARTICLE provide models with which to interpret high-resolution experiments 33,34 ; it would allow the results obtained from studies of nascent chains attached to ribosomes arrested in the process of protein synthesis to be utilized to predict nascent protein behaviour during continuous translation 13 ; it would offer insights into codon usage bias across the transcriptomes of different organisms 45 ; and it would provide a better understanding of the variety of folding and misfolding events that are possible during continuous translation 1 .…”

An analysis of NMR sensitivity enhancements obtained using non-uniform weighted sampling, and the application to protein NMR

“…We note however that small SNR improvements can translate into more substantial reductions in acquisition time. When coupled with rapid recycling experiments such as the SOFAST-HMQC, we expect that these methods may be of utility for the study of challenging and sensitivity-limited samples, such as solid-state NMR [17], solution-state NMR of proteins or metabolites at natural abundance [14,22,23], or large, dilute systems such as ribosome nascent-chain complexes [24,25]. 1.11 1.22 Table 1: Calculation and comparison of the relative sensitivities of uniform and non-uniform weighted sampling for three commonly applied window functions, w(t), computed for a signal, s(t), with a relaxation constant T 2 and observed for an acquisition time T aq , expressed in terms of the dimensionless ratio τ = T aq /T 2 .…”

Quantitative approaches to defining normal and aberrant protein homeostasis

“…[35][36][37][38] (2) New ideas about protein aggregation, 10,28 including the finding that the ability to assemble into stable and highly organised structures (e.g. amyloid fibrils) is not an unusual feature exhibited by a small group of peptides and proteins with special sequence or structural properties, but rather a property shared by most, if not all, proteins; (3) The discovery that specific aspects of protein behaviour, including their aggregation propensities 21,23,39,40 and the cellular toxicity associated with the aggregation process, 24,41 can be predicted with a remarkable degree of accuracy from the knowledge of their amino acid sequences; (4) The realisation that a wide variety of techniques originally devised for applications in nanotechnology can be used to probe the nature of protein aggregation and assembly and of the structures that emerge; 30,[42][43][44] and (5) The development of powerful approaches using model organisms for probing the origins and progression of misfolding diseases by linking concepts and principles emerging from in vitro studies to in vivo phenomena such as neurodegeneration.…”