Slow Transition Path Times Reveal a Complex Folding Barrier in a Designed Protein.

Alexander Mehlich,Johannes Stigler,Benjamin Pelz,Jie Fang,Hongbin Li

doi:10.3389/fchem.2020.587824

Abstract

De-novo designed proteins have received wide interest as potential platforms for nano-engineering and biomedicine. While much work is being done in the design of thermodynamically stable proteins, the folding process of artificially designed proteins is not well-studied. Here we used single-molecule force spectroscopy by optical tweezers to study the folding of ROSS, a de-novo designed 2x2 Rossmann fold. We measured a barrier crossing time in the millisecond range, much slower than what has been reported for other systems. While long transition times can be explained by barrier roughness or slow diffusion, we show that isotropic roughness cannot explain the measured transition path time distribution. Instead, this study shows that the slow barrier crossing of ROSS is caused by the population of three short-lived high-energy intermediates. In addition, we identify incomplete and off-pathway folding events with different barrier crossing dynamics. Our results hint at the presence of a complex transition barrier that may be a common feature of many artificially designed proteins.

Highlights

The protein-folding problem has fascinated scientists for more than half a century (Anfinsen et al, 1961)
Besides single-molecule Förster resonance energy transfer (Borgia et al, 2012; Chung et al, 2012; Soranno et al, 2012; Chung and Eaton, 2013), another well-established tool to measure the folding of individual proteins is single-molecule force spectroscopy (SMFS), where force can act as both a denaturant and a readout of protein conformational changes (Rief et al, 1997; Junker et al, 2009; Gebhardt et al, 2010; Yu et al, 2012)
We characterize the timescales of folding and barrier crossing of one of the first fully designed artificial proteins with a topology that is abundantly found in nature, the 2x2 Rossmann fold, which can serve as a scaffold for designed enzymes (Koga et al, 2012)

Summary

INTRODUCTION

The protein-folding problem has fascinated scientists for more than half a century (Anfinsen et al, 1961). To learn and understand more about the reaction that takes place when an unfolded polypeptide chain tries to “find” its correctly folded protein structure, the concept of energy landscapes is a powerful theoretical framework (Onuchic et al, 1997). Within this framework, three key parameters govern the typical timescales of an observed reaction: the height of a free energy barrier, which needs to be overcome, the curvature or stiffness at the top of that barrier, and the diffusion coefficient (Hänggi et al, 1990). We characterize the timescales of folding and barrier crossing of one of the first fully designed artificial proteins with a topology that is abundantly found in nature, the 2x2 Rossmann fold, which can serve as a scaffold for designed enzymes (Koga et al, 2012)

MATERIALS AND METHODS

Experiments of an Artificial Protein

A Barrier Harboring High-Energy

DISCUSSION

DATA AVAILABILITY STATEMENT