The Effect of tensile stress on the conformational free energy landscape of disulfide bonds

Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C-C-S-S dihedrals, and . Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force-clamp spectroscopy and computer simulation. The and angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so-called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C-C-S-S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two -carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides

Computational approaches to the calculation of spectroscopic, structural and mechanical properties of polysaccharide chains : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Massey University

In this thesis atomistic, statistical mechanical and coarse grained simulation techniques are used to study the properties of biopolymers and in particular the plant polysaccharide pectin. Spectroscopic aspects, structural and conformational behavior, and mechanical properties of the molecule in di erent physical states are addressed. After an introduction to the area and the theoretical techniques utilised herein (chapter 1), chapter 2 deals with the spectroscopic characterisation of pectin. Spectra were obtained theoretically by undertaking complete energy minimisation and Hessien calculations using DFT techniques implemented in Gamess (PC & US) packages. The calculated IR absorptions of di erent pectinic species and oligomers coupled on di erent surfaces were compared with experimental results. Herein, it is con rmed that experimental FTIR studies coupled with DFT calculations can be used as an e ective tool for the characterisation of pectin, and studying chemical coupling of the biopolymer to surfaces. In chapter 3, the properties of single chain polymer systems in controlled solvent conditions were studied using Brownian dynamics simulations, motivated by the formation of secondary structure architectures in biopolymer systems. We focus on the conformational properties of the chain in the presence of an additional torsional potential. New, interesting, and biologically relevant structures were found at the single molecule scale when a torsional potential was considered in the calculations. In chapter 4, results from DFT calculations carried out on single pectin sugar molecules (lengths and the free energies) are incorporated into a statistical mechanical model of polymer stretching, in order to obtain the force-extension behaviour of a single molecule pectin. This captures a good deal of the phenomenology of the single molecule stretching behavior of pectin. Chapter 5 summarises the conclusions of the work and nally chapter 6 suggests direction for further work

Force-induced reversal of beta-eliminations: stressed disulfide bonds in alkaline solution

Understanding the impact of tensile forces on disulfide bond cleavage is not only crucial to the breaking of cross-linkers in vulcanized materials such as strained rubber, but also to the regulation of protein activity by disulfide switches. By using ab initio simulations in the condensed phase, we investigated the response of disulfide cleavage by β-elimination to mechanical stress. We reveal that the rate- determining first step of the thermal reaction, which is the abstraction of the β- proton, is insensitive to external forces. However, forces larger than about 1 nN were found to reshape the free-energy landscape of the reaction so dramatically that a second channel is created, where the order of the reaction steps is reversed, turning β-deprotonation into a barrier-free follow-up process to C−S cleavage. This transforms a slow and force-independent process with second-order kinetics into a unimolecular reaction that is greatly accelerated by mechanical forces

Unexpected mechanochemical complexity in the mechanistic scenarios of disulfide bond reduction in alkaline solution

The reduction of disulfides has broad importance in chemistry, biochemistry and materials science, in particular upon mechanochemical activation. Here, isotensional simulations disclose that strikingly different mechanisms govern disulfide cleavage depending on external force. Crucial are desolvation and resolvation processes, which directly impact on activation free energies. The preferred pathway up to moderate forces, a bimolecular SN2 attack of OH− at sulfur, competes with unimolecular C-S bond rupture at about 2 nN, while the latter even becomes barrierless beyond. Moreover, our study brings to light a surprisingly rich reactivity scenario that includes the transformation of SN2 pathways into pure bond breaking pathways at forces within the range of 1.2 to 2.2 nN. Given that these forces are easily reached in experiments, these insights will fundamentally change our understanding of mechanochemical activation in general, which is now expected to be considerably more intricate than previously thought

Statistical analysis of the conformational diversity of native protein disulfides.

<p>Scatter plot of the / correlation for TDi (open green diamonds), DO (open indigo triangles), interchain Ig (small red circles) protein disulfides and intrachain Ig (small black circles) disulfides based on 40, 27, 69 and 927 X–ray crystal structures, respectively, obtained from the data reported in Ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108812#pone.0108812-Wong2" target="_blank">[59]</a> that are based on analyzing the Protein Data Bank.</p

Conformational diversity of the polypeptide model depending on tensile force.

<p>Probability distribution functions obtained from Boltzmann–inversion of the two–dimensional free energy landscapes from metadynamics simulations for the polypeptide model at zero force and 0.3, 0.6, and 1.0 nN in panels a to d, respectively.</p

The Effect of Tensile Stress on the Conformational Free Energy Landscape of Disulfide Bonds

<div><p>Disulfide bridges are no longer considered to merely stabilize protein structure, but are increasingly recognized to play a functional role in many regulatory biomolecular processes. Recent studies have uncovered that the redox activity of native disulfides depends on their C–C–S–S dihedrals, and . Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force–clamp spectroscopy and computer simulation. The and angles have been found to change from conformations that are open to nucleophilic attack to sterically hindered, so–called closed states upon exerting tensile stress. In view of the growing evidence of the importance of C–C–S–S dihedrals in tuning the reactivity of disulfides, here we present a systematic study of the conformational diversity of disulfides as a function of tensile stress. With the help of force-clamp metadynamics simulations, we show that tensile stress brings about a large stabilization of the closed conformers, thereby giving rise to drastic changes in the conformational free energy landscape of disulfides. Statistical analysis shows that native TDi, DO and interchain Ig protein disulfides prefer open conformations, whereas the intrachain disulfide bridges in Ig proteins favor closed conformations. Correlating mechanical stress with the distance between the two –carbons of the disulfide moiety reveals that the strain of intrachain Ig protein disulfides corresponds to a mechanical activation of about 100 pN. Such mechanical activation leads to a severalfold increase of the rate of the elementary redox reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides.</p></div

Stress–strain relation of disulfide bonds parameterized by the response of the C_α–C_α distance to tensile force.

<p>Dependence of the computed average distance between the C_α–atoms as a function of for the polypeptide model (black circles), cystine (red circles), and DEDS (green circles) depicted in Fig. 1 and obtained from force field equilibrium (at zero force) and force clamp MD (for nN) simulations. Computational reference data for DEDS obtained from QM/MM simulations are shown by brown triangles and the experimental reference based on the strained macrocycle <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108812#pone.0108812-Kucharski1" target="_blank">[27]</a> (see text) is marked by a violet square. The horizontal blue, pink and orange dotted lines are the average C_α–C_α distances of disulfide bonds in TDi, DO and interchain Ig proteins, respectively, whereas the cyan dotted line corresponds to intrachain Ig proteins; these averages have been computed using the identical data sets as those that underly Fig. 3, see caption.</p

Three models of increasing complexity used to investigate strained disulfide bonds.

<p>(a) Diethyl disulfide (DEDS), (b) cystine, and the (c) polypeptide model (see text). The collinear constant force of magnitude is applied to the terminal methyl C atoms in panel a and to N and C termini in panels b and c. The dihedral angles , and are defined in panel b.</p

Unexpected mechanochemical complexity in the mechanistic scenarios of disulfide bond reduction in alkaline solution

The reduction of disulfides has a broad importance in chemistry, biochemistry and materials science, particularly those methods that use mechanochemical activation. Here we show, using isotensional simulations, that strikingly different mechanisms govern disulfide cleavage depending on the external force. Desolvation and resolvation processes are found to be crucial, as they have a direct impact on activation free energies. The preferred pathway at moderate forces, a bimolecular S(N)2 attack of OH-at sulfur, competes with unimolecular C-S bond rupture at about 2 nN, and the latter even becomes barrierless at greater applied forces. Moreover, our study unveils a surprisingly rich reactivity scenario that also includes the transformation of concerted S(N)2 reactions into pure bond-breaking processes at specific forces. Given that these forces are easily reached in experiments, these insights will fundamentally change our understanding of mechanochemical activation in general, which is now expected to be considerably more intricate than previously thought