In silico study of osmolytic effects of choline-O-sulfate on urea induced unfolding of Trp-cage mini-protein: An atomistic view from replica exchange molecular dynamics simulation.

The event of protein folding is associated with essential biological functionalities and unfolding of protein native state can cause intra-cellular toxicity leading to biological dysfunctions and even cell death. The present study discusses the folding-unfolding equilibrium of the small globular protein Trp-cage in presence of denaturing and protecting osmolytes urea and choline-O-sulfate (COS), respectively, employing Replica Exchange Molecular Dynamics (REMD), extensive free energy calculations and temperature scanned free energy landscapes. It is shown that, while 6 M urea quite easily denatures the protein, 0.5 M and 1 M COS is able to protect the protein from urea induced denaturation at room temperature. However, REMD simulations reveal that while the protein in pure water can withstand a simulation temperature as high as 420 K without melting, the protecting effect of 0.5 M and 1 M COS is operative up to 300 and 340 K respectively. This study furnishes evidences to shed light into the protecting mechanism of COS regarding urea induced protein unfolding, thereby putting forward the use of COS as a proper protecting osmolyte towards different types of proteins.

Temperature-pressure shuffling outlier flooding method enhances the conformational sampling of proteins.

Outlier flooding method (OFLOOD) is an efficient conformational sampling method developed by the authors. In the present study, to further enhance the conformational sampling efficiency, a set of parameters (temperatures and pressures) specified as inputs in the original OFLOOD were shuffled before restarting the short-time molecular dynamics (MD) simulations. Because of the diversity of these parameters, it was confirmed that the extended OFLOOD becomes superior to the original one in finding the folding pathways of Trp-cage. © 2019 Wiley Periodicals, Inc.

Reversing the typical pH stability profile of the Trp-cage.

The Trp-cage, an 18-20 residue miniprotein, has emerged as a primary test system for evaluating computational fold prediction and folding rate determination efforts. As it turns out, a number of stabilizing interactions in the Trp-cage folded state have a strong pH dependence; all prior Trp-cage mutants have been destabilized under carboxylate-protonating conditions. Notable among the pH dependent stabilizing interactions within the Trp-cage are: (1) an Asp as the helix N-cap, (2) an H-bonded Asp9/Arg16 salt bridge, (3) an interaction between the chain termini which are in close spatial proximity, and (4) additional side chain interactions with Asp9. In the present study, we have prepared Trp-cage species that are significantly more stable at pH 2.5 (rather than 7) and quantitated the contribution of each interaction listed above. The Trp-cage structure remains constant with the pH change. The study has also provided measures of the stabilizing contribution of indole ring shielding from surface exposure and the destabilizing effects of an ionized Asp at the C-terminus of an α-helix.

Computational investigation of cold denaturation in the Trp-cage miniprotein.

The functional native states of globular proteins become unstable at low temperatures, resulting in cold unfolding and impairment of normal biological function. Fundamental understanding of this phenomenon is essential to rationalizing the evolution of freeze-tolerant organisms and developing improved strategies for long-term preservation of biological materials. We present fully atomistic simulations of cold denaturation of an α-helical protein, the widely studied Trp-cage miniprotein. In contrast to the significant destabilization of the folded structure at high temperatures, Trp-cage cold denatures at 210 K into a compact, partially folded state; major elements of the secondary structure, including the α-helix, are conserved, but the salt bridge between aspartic acid and arginine is lost. The stability of Trp-cage's α-helix at low temperatures suggests a possible evolutionary explanation for the prevalence of such structures in antifreeze peptides produced by cold-weather species, such as Arctic char. Although the 310-helix is observed at cold conditions, its position is shifted toward Trp-cage's C-terminus. This shift is accompanied by intrusion of water into Trp-cage's interior and the hydration of buried hydrophobic residues. However, our calculations also show that the dominant contribution to the favorable energetics of low-temperature unfolding of Trp-cage comes from the hydration of hydrophilic residues.

Protein Stability and Unfolding Following Glycine Radical Formation.

Glycine (Gly) residues are particularly susceptible to hydrogen abstraction; which results in the formation of the capto-dative stabilized Cα-centered Gly radical (GLR) on the protein backbone. We examined the effect of GLR formation on the structure of the Trp cage; tryptophan zipper; and the villin headpiece; three fast-folding and stable miniproteins; using all-atom (OPLS-AA) molecular dynamics simulations. Radicalization changes the conformation of the GLR residue and affects both neighboring residues but did not affect the stability of the Trp zipper. The stability of helices away from the radical center in villin were also affected by radicalization; and GLR in place of Gly15 caused the Trp cage to unfold within 1 µs. These results provide new evidence on the destabilizing effects of protein oxidation by reactive oxygen species.

Towards fast, rigorous and efficient conformational sampling of biomolecules: Advances in accelerated molecular dynamics.

BACKGROUND: Accelerated molecular dynamics (aMD) has been proven to be a powerful biasing method for enhanced sampling of biomolecular conformations on general-purpose computational platforms. Biologically important long timescale events that are beyond the reach of standard molecular dynamics can be accessed without losing the detailed atomistic description of the system in aMD. Over other biasing methods, aMD offers the advantages of tuning the level of acceleration to access the desired timescale without any advance knowledge of the reaction coordinate. SCOPE OF REVIEW: Recent advances in the implementation of aMD and its applications to small peptides and biological macromolecules are reviewed here along with a brief account of all the aMD variants introduced in the last decade. MAJOR CONCLUSIONS: In comparison to the original implementation of aMD, the recent variant in which all the rotatable dihedral angles are accelerated (RaMD) exhibits faster convergence rates and significant improvement in statistical accuracy of retrieved thermodynamic properties. RaMD in conjunction with accelerating diffusive degrees of freedom, i.e. dual boosting, has been rigorously tested for the most difficult conformational sampling problem, protein folding. It has been shown that RaMD with dual boosting is capable of efficiently sampling multiple folding and unfolding events in small fast folding proteins. GENERAL SIGNIFICANCE: RaMD with the dual boost approach opens exciting possibilities for sampling multiple timescales in biomolecules. While equilibrium properties can be recovered satisfactorily from aMD-based methods, directly obtaining dynamics and kinetic rates for larger systems presents a future challenge. This article is part of a Special Issue entitled Recent developments of molecular dynamics.

Artificial proteins as allosteric modulators of PDZ3 and SH3 in two-domain constructs: A computational characterization of novel chimeric proteins.

Artificial multidomain proteins with enhanced structural and functional properties can be utilized in a broad spectrum of applications. The design of chimeric fusion proteins utilizing protein domains or one-domain miniproteins as building blocks is an important advancement for the creation of new biomolecules for biotechnology and medical applications. However, computational studies to describe in detail the dynamics and geometry properties of two-domain constructs made from structurally and functionally different proteins are lacking. Here, we tested an in silico design strategy using all-atom explicit solvent molecular dynamics simulations. The well-characterized PDZ3 and SH3 domains of human zonula occludens (ZO-1) (3TSZ), along with 5 artificial domains and 2 types of molecular linkers, were selected to construct chimeric two-domain molecules. The influence of the artificial domains on the structure and dynamics of the PDZ3 and SH3 domains was determined using a range of analyses. We conclude that the artificial domains can function as allosteric modulators of the PDZ3 and SH3 domains. Proteins 2016; 84:1358-1374. © 2016 Wiley Periodicals, Inc.

FF12MC: A revised AMBER forcefield and new protein simulation protocol.

Specialized to simulate proteins in molecular dynamics (MD) simulations with explicit solvation, FF12MC is a combination of a new protein simulation protocol employing uniformly reduced atomic masses by tenfold and a revised AMBER forcefield FF99 with (i) shortened CH bonds, (ii) removal of torsions involving a nonperipheral sp(3) atom, and (iii) reduced 1-4 interaction scaling factors of torsions ϕ and ψ. This article reports that in multiple, distinct, independent, unrestricted, unbiased, isobaric-isothermal, and classical MD simulations FF12MC can (i) simulate the experimentally observed flipping between left- and right-handed configurations for C14-C38 of BPTI in solution, (ii) autonomously fold chignolin, CLN025, and Trp-cage with folding times that agree with the experimental values, (iii) simulate subsequent unfolding and refolding of these miniproteins, and (iv) achieve a robust Z score of 1.33 for refining protein models TMR01, TMR04, and TMR07. By comparison, the latest general-purpose AMBER forcefield FF14SB locks the C14-C38 bond to the right-handed configuration in solution under the same protein simulation conditions. Statistical survival analysis shows that FF12MC folds chignolin and CLN025 in isobaric-isothermal MD simulations 2-4 times faster than FF14SB under the same protein simulation conditions. These results suggest that FF12MC may be used for protein simulations to study kinetics and thermodynamics of miniprotein folding as well as protein structure and dynamics. Proteins 2016; 84:1490-1516. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.

A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

Folding-unfolding asymmetry and a RetroFold computational algorithm.

We treat protein folding as molecular self-assembly, while unfolding is viewed as disassembly. Fracture is typically a much faster process than self-assembly. Self-assembly is often an exponentially decaying process, since energy relaxes due to dissipation, while fracture is a constant-rate process as the driving force is opposed by damping. Protein folding takes two orders of magnitude longer than unfolding. We suggest a mathematical transformation of variables, which makes it possible to view self-assembly as time-reversed disassembly, thus folding can be studied as reversed unfolding. We investigate the molecular dynamics modelling of folding and unfolding of the short Trp-cage protein. Folding time constitutes about 800 ns, while unfolding (denaturation) takes only about 5.0 ns and, therefore, fewer computational resources are needed for its simulation. This RetroFold approach can be used for the design of a novel computation algorithm, which, while approximate, is less time-consuming than traditional folding algorithms.

Topological bio-scaling analysis as a universal measure of protein folding.

Scaling relationships for polymeric molecules establish power law dependencies between the number of molecular segments and linear dimensions, such as the radius of gyration. They also establish spatial topological properties of the chains, such as their dimensionality. In the spatial domain, power exponents α = 1 (linear stretched molecule), α = 0.5 (the ideal chain) and α = 0.333 (compact globule) are significant. During folding, the molecule undergoes the transition from the one-dimensional linear to the three-dimensional globular state within a very short time. However, intermediate states with fractional dimensions can be stabilized by modifying the solubility (e.g. by changing the solution temperature). Topological properties, such as dimension, correlate with the interaction energy, and thus by tuning the solubility one can control molecular interaction. We investigate these correlations using the example of a well-studied short model of Trp-cage protein. The radius of gyration is used to estimate the fractal dimension of the chain at different stages of folding. It is expected that the same principle is applicable to much larger molecules and that topological (dimensional) characteristics can provide insights into molecular folding and interactions.

Application of a Distance-Dependent Sigmoidal Dielectric Constant to the REMC/SAAP3D Simulations of Chignolin, Trp-Cage, and the G10q Mutant.

The replica-exchange Monte Carlo method based on the single amino acid potential (SAAP) force field, i.e., REMC/SAAP3D, was recently developed by our group for the molecular simulation of short peptides. In this study, the method has been improved by applying a distance-dependent dielectric (DDD) constant and extended to the peptides containing D-amino acid (AA) residues. For chignolin (10 AAs), a sigmoidal DDD model reasonably allocated the native-like ß-hairpin structure with all-atom root mean square deviation (RMSD) = 2.0 Å as a global energy minimum. The optimal DDD condition was subsequently applied for Trp-cage (20 AAs) and its G10q mutant. The native-like α-rich folded structures with main-chain RMSD = 3.7 and 3.8 Å were obtained as global energy minima for Trp-cage and G10q, respectively. The results suggested that the REMC/SAAP3D method with the sigmoidal DDD model is useful for structural prediction for the short peptides comprised of up to 20 AAs. In addition, the relative contributions of SAAP to the total energy (%SAAP) were evaluated by energetic component analysis. The ratios of %SAAP were about 40 and 20% for chignolin and Trp-cage (or G10q), respectively. It was proposed that SAAP is more important for the secondary structure formation than for the assembly to a higher-order folded structure, in which the attractive van der Waals interaction may play a more important role.

Itinerary profiling to analyze a large number of protein-folding trajectories.

Understanding how proteins fold through a vast number of unfolded states is a major subject in the study of protein folding. Herein, we present itinerary profiling as a simple method to analyze molecular dynamics trajectories, and apply this method to Trp-cage. In itinerary profiling, structural clusters included in a trajectory are represented by a bit sequence, and a number of trajectories, as well as the structural clusters, can be compared and classified. As a consequence, the structural clusters that characterize the foldability of trajectories were able to be identified. The connections between the clusters were then illustrated as a network and the structural features of the clusters were examined. We found that in the true folding funnel, Trp-cage formed a left-handed main-chain topology and the Trp6 side-chain was located at the front of the main-chain ring, even in the initial unfolded states. In contrast, in the false folding funnel of the pseudo-native states, in which the Trp6 side-chain is upside down in the protein core, Trp-cage had a right-handed main-chain topology and the Trp side-chain was at the back. The initial topological partition, as determined by the main-chain handedness and the location of the Trp residue, predetermines Trp-cage foldability and the destination of the trajectory to the native state or the pseudo-native states.

Correlation analysis for heat denaturation of Trp-cage miniprotein with explicit solvent.

Energetics was analyzed for Trp-cage miniprotein in water to elucidate the solvation effect in heat denaturation. The solvation free energy was computed for a set of protein structures at room and high temperatures with all-atom molecular dynamics simulation combined with the solution theory in the energy representation, and its correlations were investigated against the intramolecular (structural) energy of the protein and the average interaction energy of the protein with the solvent water. It was observed both at room and high temperatures that the solvation free energy is anticorrelated to the structural energy and varies in parallel to the electrostatic component of the protein-water interaction energy without correlations to the van der Waals and excluded-volume components. When the set of folded structures sampled at room temperature was compared with the set of unfolded ones at high temperature, it was found that the preference order of the two sets is in correspondence to the van der Waals and excluded-volume components in the sum of the protein intramolecular and protein-water intermolecular interactions and is not distinguished by the electrostatic component.

Influence of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage.

We examine the effect of the ionic liquid [C4mpy][Tf2N] on the structure of the miniprotein Trp-cage and contrast these results with the behavior of Trp-cage in water. We find the ionic liquid has a dramatic effect on Trp-cage, though many similarities with aqueous Trp-cage are observed. We assess Trp-cage folding by monitoring root mean square deviation from the crystallographic structure, radius of gyration, proline cis/trans isomerization state, protein secondary structure, amino acid contact formation and distance, and native and non-native contact formation. Starting from an unfolded configuration, Trp-cage folds in water at 298 K in less than 500 ns of simulation, but has very little mobility in the ionic liquid at the same temperature, which can be ascribed to the higher ionic liquid viscosity. At 365 K, the mobility of the ionic liquid is increased and initial stages of Trp-cage folding are observed, however Trp-cage does not reach the native folded state in 2 µs of simulation in the ionic liquid. Therefore, in addition to conventional molecular dynamics, we also employ scaled molecular dynamics to expedite sampling, and we demonstrate that Trp-cage in the ionic liquid does closely approach the aqueous folded state. Interestingly, while the reduced mobility of the ionic liquid is found to restrict Trp-cage motion, the ionic liquid does facilitate proline cis/trans isomerization events that are not seen in our aqueous simulations.

Achieving Rigorous Accelerated Conformational Sampling in Explicit Solvent.

Molecular dynamics simulations can provide valuable atomistic insights into biomolecular function. However, the accuracy of molecular simulations on general-purpose computers depends on the time scale of the events of interest. Advanced simulation methods, such as accelerated molecular dynamics, have shown tremendous promise in sampling the conformational dynamics of biomolecules, where standard molecular dynamics simulations are nonergodic. Here we present a sampling method based on accelerated molecular dynamics in which rotatable dihedral angles and nonbonded interactions are boosted separately. This method (RaMD-db) is a different implementation of the dual-boost accelerated molecular dynamics, introduced earlier. The advantage is that this method speeds up sampling of the conformational space of biomolecules in explicit solvent, as the degrees of freedom most relevant for conformational transitions are accelerated. We tested RaMD-db on one of the most difficult sampling problems - protein folding. Starting from fully extended polypeptide chains, two fast folding α-helical proteins (Trpcage and the double mutant of C-terminal fragment of Villin headpiece) and a designed ß-hairpin (Chignolin) were completely folded to their native structures in very short simulation time. Multiple folding/unfolding transitions could be observed in a single trajectory. Our results show that RaMD-db is a promisingly fast and efficient sampling method for conformational transitions in explicit solvent. RaMD-db thus opens new avenues for understanding biomolecular self-assembly and functional dynamics occurring on long time and length scales.

Maximum probability reaction sequences in stochastic chemical kinetic systems.

The detailed behavior of many molecular processes in the cell, such as protein folding, protein complex assembly, and gene regulation, transcription and translation, can often be accurately captured by stochastic chemical kinetic models. We investigate a novel computational problem involving these models - that of finding the most-probable sequence of reactions that connects two or more states of the system observed at different times. We describe an efficient method for computing the probability of a given reaction sequence, but argue that computing most-probable reaction sequences is EXPSPACE-hard. We develop exact (exhaustive) and approximate algorithms for finding most-probable reaction sequences. We evaluate these methods on test problems relating to a recently-proposed stochastic model of folding of the Trp-cage peptide. Our results provide new computational tools for analyzing stochastic chemical models, and demonstrate their utility in illuminating the behavior of real-world systems.