Measurement of energy landscape roughness of folded and unfolded proteins

Abstract

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4-5 k B T). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 k B T) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues. photochemical trigger | subdiffusion M ajor advances have been made in recent years in understanding dynamic aspects of protein conformational changes, particularly protein folding; however, many issues remain to be solved (1). Among these are the properties of the unfolded protein ensemble and the role of residual structure of denatured proteins in promoting folding (2), the heterogeneity of microscopic folding pathways (3), and the existence of multiple distinct, but only transiently populated, intermediates (4). Particularly for fast-folding proteins, the idea of downhill folding, i.e., the absence of a significant barrier, has been suggested as an alternative mechanism (5, 6), but it is not clear to what extent fast-folding proteins make use of this mechanism. On the other hand, technical progress has made it possible to observe multiple folding and unfolding events in millisecond all-atom molecular dynamics simulations. Such simulations have shown that some proteins always follow the same folding pathway, whereas others have several different pathways (7). Moreover, individual folding events occur with submicrosecond transit times through a distinct transition state but are separated by long waiting times, which yield the experimentally observed folding times (8).The idea of motion on a rugged energy landscape (9-11) has been used widely to describe conformational changes in protei...

The roughness of the protein energy landscape results in anomalous diffusion of the polypeptide backbone

“…Similar results were found for a wide range of parameters. 32 Thus, the experimentally observed power law time dependence k inst (t) B t À0.94 over nine orders of magnitude in time can be reproduced almost perfectly with simulations assuming a wide range of local trapping times which leads to subdiffusive motion of the peptide segments to which the radicals are bound, whereas normal diffusion is not able to reproduce this power dependence even when accounting for the tethering, chain stiffness and excluded volume effects of the polypeptide backbone. This provides compelling evidence that the relative motion of polypeptide backbone segments is strongly subdiffusive, with a subdiffusional parameter a of around 0.3.…”

“…We then describe a recently reported alternative approach for the experimental characterisation of backbone dynamics and energy landscape roughness. 32 Most interestingly, these results show that the polypeptide backbone does not follow normal diffusional behaviour, but undergoes subdiffusional motion due to a wide range of trapping times arising from the hierarchy of local energy minima on the rough energy landscape. Analysis of the experimental observations allows a quantitative determination of the roughness.…”

Speed of Conformational Change: Comparing Explicit and Implicit Solvent Molecular Dynamics Simulations

“…Biophysical Journal 108(5) 1153-1164 perhaps even artificially so compared to explicit solvent (53) and, possibly, reality (95).…”

Effects of pressure on the dynamics of an oligomeric protein from deep-sea hyperthermophile

“…These lead to the concept of a multidimensional potential energy landscape (EL) that specifies a complete description of CSs in proteins (6)(7)(8)(9). The existence of an EL was proposed by H. Frauenfelder and others in the 1970s and has been validated both by computations and by experiments (6)(7)(8)(9)(10)(11)(12).…”