Atomic scale insights into urea-peptide interactions in solution.

Nicola Steinke,Luis Carlos Pardo,Christian D Lorenz,Sylvia E Mclain,Richard J Gillams

doi:10.1039/c5cp06646h

Abstract

The mechanism by which proteins are denatured by urea is still not well understood, especially on the atomic scale where these interactions occur in vivo. In this study, the structure of the peptide GPG has been investigated in aqueous urea solutions in order to understand the combination of roles that both urea and water play in protein unfolding. Using a combination of neutron diffraction enhanced by isotopic substitution and computer simulations, it was found, in opposition with previous simulations studies, that urea is preferred over water around polar and charged portions of the peptides. Further, it appears that while urea directly replaces water around the nitrogen groups on GPG that urea and water occupy different positions around the peptide bond carbonyl groups. This suggests that urea may in fact weaken the peptide bond, disrupting the peptide backbone, thus ultimately causing denaturation.

Highlights

It has long been known that proteins unfold in the presence of urea in vitro.[1]
See DOI: 10.1039/ c5cp06646h proposed, where Hua et al have suggested that once urea has expelled the first hydration layer around the protein, the hydrophobic core is penetrated by urea as opposed to water.[7,8]. This is contrary to other investigations which found that the hydrophobic core was preferentially solvated by water in the first instance.[9]. Despite this relatively wide range of computational studies, there is little experimental information concerning how urea molecules interact with different components of proteins on the atomic scale as these interactions can only be probed via techniques which measure on the order of angstroms (10À10 m Å)
The corresponding Fourier transformations which show the data in real space are shown in the Electronic supplementary information (ESI),† along with the Empirical potential structure refinement (EPSR) fits and the MD comparison

Summary

Introduction

It has long been known that proteins unfold in the presence of urea in vitro.[1]. There are a multiplicity of theories as to how the presence of this small molecule denatures these relatively large macromolecules, yet there is no consensus on the mechanism by which this occurs. One proposed mechanism for this is that urea indirectly ‘‘dries’’ the protein lowering the protein hydration and weakening the hydrophobic effect.[2]. Despite this relatively wide range of computational studies, there is little experimental information concerning how urea molecules interact with different components of proteins on the atomic scale as these interactions can only be probed via techniques which measure on the order of angstroms (10À10 m Å). While NMR does provide some experimental evidence of folded and unfolded protein structure in aqueous solution upon the addition of urea, the timescale of NMR makes it difficult to assess the process of unfolding, direct urea–protein interactions and more importantly how urea and water molecules might or might not interact with each other to affect a structural change in the protein.

Methods

Results

Conclusion