Biology subjects

Marqusee, S.

Publications and source records attributed to Marqusee, S..

3 recordsLinked to original sources

bioRxiv2018

Modulating long-range energetics via helix stabilization: a case study using T4 lysozyme

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization.

biophysics↗

bioRxiv2018

A small single-domain protein folds through the same pathway on- and off- the ribosome

In vivo, proteins fold and function in a complex environment where they are subject to many stresses that can modulate protein energy landscapes. One aspect of the environment pertinent to protein folding is the ribosome, since proteins have the opportunity to fold while still bound to the ribosome during translation. We use a combination of force and chemical denaturant (chemo-mechanical unfolding), as well as point mutations, to characterize the folding mechanism of the src SH3 domain both as a stalled ribosome nascent chain and free in solution. Our results indicate that src SH3 folds through the same pathway on and off the ribosome. Molecular simulations also indicate that the ribosome does not affect the folding pathway for this small protein. Taken together, we conclude that the ribosome does not alter the folding mechanism of this small protein, which appears to fold at the mouth of the ribosome as the protein emerges from the exit tunnel. These results, if general, suggest the ribosome may exert a bigger influence on the folding of multi-domain proteins or protein domains that can partially fold before the entire domain sequence is outside the ribosome exit tunnel.

biophysics↗

bioRxiv2018

Tracing a proteins folding pathway over evolutionary time using ancestral sequence reconstruction and hydrogen exchange

The conformations populated during protein folding have been studied for decades; yet, their evolutionary importance remains largely unexplored. Ancestral sequence reconstruction allows access to proteins across evolutionary time, and new methods such as pulsed-labeling hydrogen exchange coupled with mass spectrometry allow determination of folding intermediate structures at near amino-acid resolution. Here, we combine these techniques to monitor the folding of the ribonuclease H family along the evolutionary lineages of T. thermophilus and E. coli RNase H. All homologs and ancestral proteins studied populate a similar folding intermediate despite being separated by billions of years of evolution. Even though this conformation is conserved, the pathway leading to it has diverged over evolutionary time, and rational mutations can alter this trajectory. Our results demonstrate that evolutionary processes can affect the energy landscape to preserve or alter specific features of a proteins folding pathway.

biochemistry↗