Insights into the Role of Side-Chain Team Work in nDsbDOx/Red Proteins: Mechanism of Substrate Binding.

N-terminal disulfide bond oxidoreductase (nDsbDOx/Red) proteins display divergent substrate binding mechanisms depending on the conformational changes to the Phe70 cap, which is also dependent on the disulfide redox state. In nDsbDOx, the cap dynamics is complex (shows both open/closed Phe70 cap conformations), resulting in an active site that is highly flexible. So the system's active site is conformationally selective (the active site adapts before substrate binding) toward its substrate. In nDsbDRed, the cap is generally closed, resulting in induced fit-type binding (adapts after substrate approach). Recent studies predict Tyr40 and Tyr42 residues to act as internal nucleophiles (Tyr40/42O-) for disulfide association/dissociation in nDsbDOx/Red, supplementing the electron transfer channel. From this perspective, we investigate the cap dynamics and the subsequent substrate binding modes in these proteins. Our molecular dynamics simulations show that the cap opening eliminates Tyr42O- electrostatic interactions irrespective of the disulfide redox state. The active site becomes highly flexible, and the conformational selection mechanism governs. However, Tyr40O- formation does not alter the chemical environment; the cap remains mostly closed and plausibly follows the induced fit mechanism. Thus, it is apparent that mostly Tyr42O- facilitates the internal nucleophile-mediated self-preparation of nDsbDOx/Red proteins for binding.

Superoxide to Peroxide Interconversion in Ni-TMC Complexes: The Significance of Structure and Spin States.

A deeper comprehension of the characteristics of metal-superoxide and metal-peroxide chemical species is imperative, considering their pivotal functions in oxygen transport, enzymatic activation, and catalytic oxygenations. O2 activation is mediated by the interconversion of superoxide and peroxide species. Even though there are multiple studies on metal-superoxide and -peroxide intermediates, robust examples of their interconversion processes are scarce synthetically. For example, Ni-superoxide/peroxide complexes have been characterized with N-Tetramethylated Cyclam (TMC) ligands with different ring sizes, i.e., Nickel(II)-superoxide complex is characterized with 14-TMC while Nickel(III)-peroxide complex with 12-TMC. Later, both complexes were obtained with 13-TMC ligand by employing different bases; interestingly, no evidence of interconversion between them was identified. What are the factors influencing these processes and why is this preference? We attempt a computational analysis of this issue and provide arguments based on our conclusions. 2-dimensional potential energy scan is performed on the 12-TMC, 13-TMC, and 14-TMC systems to identify the reaction path connecting superoxide and peroxide species. Analyses indicate that structure and spin states play a significant role in determining the probability of interconversion. The superoxide-peroxide interconversion process appears to be bound by their propensity for distinct structural features and spin states.

Exceptional stability of ultrasmall cubic copper metal nanoclusters - a molecular dynamics study.

The fabrication of shape-selective coinage metal nanoclusters (MNCs) has promising applications due to their exceptional physical and chemical molecule-like properties. However, the stability of the specific geometry of the nanoclusters, such as their cubic shapes, is unclear and has been unraveled by assessing the nanoclusters' interactions with different environments. In this work, we investigate the morphological stability of cubic structured, coinage metal nanoclusters of varying sizes ranging from 14 to 1099 atoms. The impact of solvent environments like water and the presence of ionic liquids (IL) on the stabilization of the MNCs were assessed using molecular dynamics (MD) simulations. In general, smaller MNCs composed of less than 256 atoms encountered structural distortion easily compared to the larger ones, which preserved their cubic morphology with minimal surface aberrations in water. However, in the presence of 4M 1-butyl-1,1,1-trimethyl ammonium methane sulfonate [N1114][C1SO3] IL solution, the overall cubic shape of the MNCs was successfully preserved. Strikingly, it is observed that in contrast to the noble MNCs like Au and Ag, the cubic morphology for Cu MNCs with sizes less than 256 atoms exhibited significant stability even in the absence of IL.

Chemical Tailoring Assisted non-TADF to TADF Switching in Carbazole-Benzophenone Emitter - An In-silico Investigation.

Organic light-emitting diodes (OLEDs) have become one of the most popular lighting technologies since they offer several advantages over conventional devices. In carbazole-benzophenone (CzBP) OLED devices, the polymeric form of the compound is previously reported to be Thermally Activated Delayed Fluorescence (TADF)-active (ΔEST ≈0.12 eV), while the monomer (CzBP) (ΔEST ≈0.39 eV) does not. The present study examines the effect of chemical tailoring on the optical and photophysical properties of CzBP using DFT and TDDFT methods. The introduction of a single -NO2 group or di-substitution (-NO2 , -COOH or -CN) in the selected LUMO region of the reference CzBP monomer significantly reduces ΔEST ≈0.01 eV, projecting these systems as potential TADF-active emitters. Furthermore, the chemical modification of CzBP-LUMO alters the two-step TADF mechanism (T1 →T2 →S1 ) in CzBP (ES1 >ET2 >ET1 ) to the Direct Singlet Harvesting (T1 →S1 ) mechanism (ET2 >ES1 >ET1 ), which has recently been identified in the fourth-generation OLED materials.

Multifaceted folding-unfolding landscape of the TrpZip2 ß-hairpin and the role of external sub-piconewton mechanical tensions.

Proteins can experience uneven tensions of the order of tens of piconewtons when exposed to different solvent environment due to the thermal motion of the solvent. It is also true that biomolecules, especially proteins, are subjected to a variety of mechanical tensions generated by several factors, including mechanically assisted translocation and pressure gradients within living systems. Here, we use metadynamics simulations to revisit the folding-unfolding of the TrpZip2 ß-hairpin and redefine it from the perspective of an external force of a sub-piconewton magnitude acting on the ends of the hairpin. The chosen forces, while preserving the morphology of the ß-hairpin chain when it is pulled, are capable of influencing the conformational behavior of the chain during folding and unfolding. Our investigations confirm that the TrpZip2 ß-hairpin exhibits a zipper (zip-out) mechanism for folding-unfolding in both mechanically unbiased and biased (with a 30 pN end force) situations. However, it is important to note that they present marked differences in their folding and unfolding paths, with the mechanically biased system capable of becoming trapped in various intermediate states. Both unbiased and biased scenarios of the hairpin indicate that the hairpin turn is highly stable during the folding-unfolding event and initiates folding. More importantly we confirm that the existing heterogeneity in the TrpZip2 ß-hairpin folding-unfolding is a consequence of the wide range of conformations observed, owing to the different trapped intermediates caused by the uneven forces it may experience in solution.

Disulfide Isomerization in nDsbD-DsbC Complex - Exploring an Internal Nucleophile Mediated Reaction Pathway.

The disulfide bond redox chemistry of proteins is believed to be mostly governed by the proton motive force. The nucleophilic and α-elimination mechanisms are also found to supplement the formation and scission of the S-S bonds. On these grounds, the possibility for an internal nucleophile assisted disulfide bond formation in the nDsbD-DsbC complex was proposed way back. Using QM/MM MD metadynamics simulations, we explore the feasibility of the proposed mechanism. Our simulations highlight the formation of the internal nucleophile Tyr42 O- and Tyr40 O- which further generates Cys103 S- necessary for the disulfide bond formation in nDsbD. Our results illustrate how the isomerase DsbC is functionally activated by nDsbD in gram-negative bacteria. Also, we foresee that the results will be important for modelling anti-bacterial compounds based on nDsbD.

Towards solvent regulated self-activation of N-terminal disulfide bond oxidoreductase-D.

N-terminal disulfide bond oxidoreductase-D (nDsbD), an essential redox enzyme in Gram-negative bacteria, consists of a single disulfide bond (Cys103-Cys109) in its active site. The enzymatic functions are believed to be regulated by an electron transfer mediated redox switching of the disulfide bond, which is vital in controlling bacterial virulence factors. In light of the disulfide bond's inclination towards nucleophilic cleavage, it is also plausible that an internal nucleophile could second the existing electron transfer mechanism in nDsbD. Using QM/MM MD metadynamics simulations, we explore different possibilities of generating an internal nucleophile near the nDsbD active site, which could serve as a fail-over mechanism in cleaving the disulfide bond. The simulations show the formation of the internal nucleophile Tyr42O- (F ≈ 9 kcal mol-1) and its stabilization through the solvent medium. The static gas-phase calculations show that Tyr42O- could be a potential nucleophile for cleaving the S-S bond. Most strikingly, it is also seen that Tyr42O- and Asp68OH communicate with each other through a proton-hole like water wire (F ≈ 12 kcal mol-1), thus modulating the nucleophile formation. Accordingly, we propose the role of a solvent in regulating the internal nucleophilic reactions and the subsequent self-activation of nDsbD. We believe that this could be deterministic while designing enzyme-targeted inhibitor compounds.

An account on the factors determining the extra stability of the ß-hairpin from B1 domain of protein G.

The folding-unfolding of a 16 residue polypeptide, a ß-hairpin in B1 domain of protein G is investigated here to account for the factors assisting the extra stability of the polypeptide in the presence of an explicit solvent and even when a denaturant like urea is present in the medium. It is observed here that the backbone H-bond network well defines the folded state and is even capable of forming the folded state, but it is not the only criteria for making a stable ß-hairpin fold. Factors such as the side chain H-bonds and the alignment of the certain hydrophobic group side chains play a prominent role in preserving the ß-hairpin structure and thus providing an extra stability to the hairpin architecture. It is also affirmed that the mentioned hydrophobic groups side chain interactions are very crucial in holding the ß-hairpin together and without which the hairpin collapses completely. We also confirm that the denaturant urea acts on the GB1-hairpin backbone H-bonds and in the presence of strong hydrophobic interactions with a consistent side chain H-bonding network, the denaturation being comparatively a slower process with respect to the protein devoid of the side chain interactions.Communicated by Ramaswamy H. Sarma.

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 SN2 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 SN2 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.

Force-Induced Reversal of ß-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 ß-elimination to mechanical stress. We reveal that the rate-determining first step of the thermal reaction, which is the abstraction of the ß-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 ß-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.

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, χ2 and χ'2. Moreover, the interplay of chemical reactivity and mechanical stress of disulfide switches has been recently elucidated using force-clamp spectroscopy and computer simulation. The χ2 and χ'2 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 a-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 S(N)2 reaction step. All these findings constitute a step forward towards achieving a full understanding of functional disulfides.

The Janus-faced role of external forces in mechanochemical disulfide bond cleavage.

Recent force microscopy measurements on the mechanically activated cleavage of a protein disulfide bond through reaction with hydroxide ions revealed that for forces greater than 0.5 nN, the acceleration of the reaction rate is substantially reduced. Here, using ab initio simulations, we trace this 'reactivity switch' back to a dual role played by the mechanical force, which leads to antagonistic effects. On the one hand, the force performs work on the system, and thereby accelerates the reaction. On the other hand, the force also induces a conformational distortion that involves the S-S-C-C dihedral angle, which drives the disulfide into a conformation that is shielded against nucleophilic attack because of steric hindrance. The discovery of force-induced conformational changes that steer chemical reactivity provides a new key concept that is expected to be relevant beyond this specific case, for example in understanding how 'disulfide switches' regulate protein function and for the rational design of mechanoresponsive materials.

Unraveling solvent-mediated reaction pathways leading to regiospecific mechanochemical cleavage of disulfide bonds in peptides.

Stressing disulfide bonds! Nucleophilic thiol-disulfide exchange reactions within the I27 domain of titin were previously investigated with force clamp AFM. Here, all possible pathways associated with disulfide bond scission at constant tensile force are revealed in terms of end-to-end distances by using force clamp molecular dynamics. The simulations, together with experimental data unravel the competition between mechanochemical bond activation and solvent-mediated regiospecificity exhibited during SS bond cleavage due to the nucleophilic substitution mechanism within a stretched peptide.

Langevin dynamics simulations reveal biologically relevant folds arising from the incorporation of a torsional potential.

The conformational behaviour of polymer chains has been examined using Langevin dynamics simulation techniques. Polymer chains were modelled as "beads" undergoing Brownian motion in a defined potential that accounted for stretching, bending and solvation energies. As expected, the competition between chain stiffness and solvent interactions was found to yield standard swollen or collapsed configurations in good or poor solvents, respectively. However, when a torsional term was introduced into the model, additional biologically relevant conformations such as helices, sheets, turns and hairpins naturally arose.