The Kinetics of helix unfolding: Molecular dynamics simulations with Milestoning

The unfolding process of a helical heteropeptide is studied by computer simulation in explicit solvent. A combination of a functional optimization to determine the reaction coordinate and short time trajectories between “Milestones” is used to study the kinetic of the unfolding. One hundred unfolding trajectories along three different unfolding pathways are computed between all nearby Milestones providing adequate statistics to compute the overall first passage time. The radius of gyration is smaller for intermediate configurations compared to the initial and final states suggesting that the kinetics (but not the thermodynamics) is sensitive to pressure. The transitions are dominated by local bond rotations (the ψ dihedral angle) that are assisted by significant nonmonotonic fluctuations of nearby torsions. The most effective unfolding pathway is via the N-terminal

Kinetic pathway analysis of an α-helix in two protonation states: Direct observation and optimal dimensionality reduction

This article may be downloaded for personal use only. Any other use requires prior permission of the author and AIP Publishing. This article appeared in J. Chem. Phys. 150, 074902 (2019); https://doi.org/10.1063/1.5082192 and may be found at https://aip.scitation.org/doi/full/10.1063/1.5082192Thermodynamically stable conformers of secondary structural elements make a stable tertiary/quaternary structure that performs its proper biological function efficiently. Formation mechanisms of secondary and tertiary/quaternary structural elements from the primary structure are driven by the kinetic properties of the respective systems. Here we have carried out thermodynamic and kinetic characterization of an alpha helical heteropeptide in two protonation states, created with the addition and removal of a proton involving a single histidine residue in the primary structure. Applying far-UV circular dichroism spectroscopy, the alpha helix is observed to be significantly more stable in the deprotonated state. Nanosecond laser temperature jump spectroscopy monitoring time-resolved tryptophan fluorescence on the protonated conformer is carried out to measure the kinetics of this system. The measured relaxation rates at a final temperature between 296K and 314 K generated a faster component of 20 ns–11 ns and a slower component of 314 ns–198 ns. Atomically detailed characterization of the helix-coil kinetic pathways is performed based on all-atom molecular dynamics trajectories of the two conformers. Application of clustering and kinetic coarse-graining with optimum dimensionality reduction produced description of the trajectories in terms of kinetic models with two to five states. These models include aggregate states corresponding to helix, coil, and intermediates. The “coil” state involves the largest number of conformations, consistent with the expected high entropy of this structural ensemble. The “helix” aggregate states are found to be mixed with the full helix and partially folded forms. The experimentally observed higher helix stability in the deprotonated form of the alpha helical heteropeptide is reflected in the nature of the “helix” aggregate state arising from the kinetic model. In the protonated form, the “coil” state exhibits the lowest free energy and longest lifetime, while in the deprotonated form, it is the “helix” that is found to be most stable. Overall, the coarse grained models suggest that the protonation of a single histidine residue in the primary structure induces significant changes in the free energy landscape and kinetic network of the studied helix-forming heteropeptide

Picosecond Time-Resolved Fourier Transform Raman Spectroscopy of 9,10-Diphenylanthracene in the Excited Singlet State

This is the publisher's version, also available electronically from http://www.opticsinfobase.org/as/abstract.cfm?URI=as-49-5-645.Time-resolved Fourier transform Raman spectroscopy of the highly fluorescent chromophore 9,10-diphenylanthracene (DPA) in cyclohexane and ethanol is described. Raman spectra of the first excited singlet state of DPA were obtained with 100-ps resolution at several time delays between pump pulses at 355 nm and probe pulses at 1064 nm. The near-infrared excited-state Raman scattering is enhanced by resonance with an excited-state transition of DPA. The excited-state Raman bands decay in about 5-6 ns. Evidence for interaction of the solvent with the DPA excited state is observed in the cyclohexane C-H stretching bands

Length Dependent Folding Kinetics of Alanine-Based Helical Peptides from Optimal Dimensionality Reduction

We present a computer simulation study of helix folding in alanine homopeptides (ALA)n of length n = 5, 8, 15, and 21 residues. Based on multi-microsecond molecular dynamics simulations at room temperature, we found helix populations and relaxation times increasing from about 6% and ~2 ns for ALA5 to about 60% and ~500 ns for ALA21, and folding free energies decreasing linearly with the increasing number of residues. The helix folding was analyzed with the Optimal Dimensionality Reduction method, yielding coarse-grained kinetic models that provided a detailed representation of the folding process. The shorter peptides, ALA5 and ALA8, tended to convert directly from coil to helix, while ALA15 and ALA21 traveled through several intermediates. Coarse-grained aggregate states representing the helix, coil, and intermediates were heterogeneous, encompassing multiple peptide conformations. The folding involved multiple pathways and interesting intermediate states were present on the folding paths, with partially formed helices, turns, and compact coils. Statistically, helix initiation was favored at both termini, and the helix was most stable in the central region. Importantly, we found the presence of underlying universal local dynamics in helical peptides with correlated transitions for neighboring hydrogen bonds. Overall, the structural and dynamical parameters extracted from the trajectories are in good agreement with experimental observables, providing microscopic insights into the complex helix folding kinetics

Experiments and comprehensive simulations of the formation of a helical turn

We consider the kinetics and thermodynamics of a helical turn formation in the peptide Ac-WAAAH-NH2. NMR measurements indicate that the peptide has significant tendency to form a structure of a helical turn, while temperature dependent CD establishes the helix fraction at different temperatures. Molecular Dynamics and Milestoning simulations agree with experimental observables and suggests an atomically detailed picture for the turn formation. Using a network representation two alternative mechanisms of folding are identified: (i) a direct cooperative mechanism from the unfolded to the folded state without intermediate formation of hydrogen bonds and (ii) an indirect mechanism with structural intermediates with two residues in a helical conformation. This picture is consistent with kinetic measurements that reveal two experimental time scales of sub nanosecond and several nanoseconds

Influence of temperature and viscosity on anthracene rotational diffusion in organic solvents: Molecular dynamics simulations and fluorescence anisotropy study

This is the publisher's version, also available electronically from http://scitation.aip.org/content/aip/journal/jcp/107/21/10.1063/1.475172.Molecular dynamics simulations and fluorescenceanisotropy decay measurements are used to investigate the rotational diffusion of anthracene in two organic solvents—cyclohexane and 2-propanol—at several temperatures. Molecular dynamics simulations of 1 ns length were performed for anthracene in cyclohexane (at 280, 296, and 310 K) and in 2-propanol (at 296 K). The calculated time constants for reorientation of the short in-plane axis were 7–9 and 11–16 ps at 296 K in cyclohexane and 2-propanol, respectively, in excellent agreement with corresponding fluorescence depolarization measurements of 8 and 14 ps. The measured rotational reorientation times and the calculated average rotational diffusion coefficients varied in accord with Debye–Stokes–Einstein theory. Their magnitudes were close to values predicted for an ellipsoid of shape and size equivalent to an anthracene molecule, and exhibited predictable variation with external conditions—increasing with temperature and decreasing with solventviscosity. However, analysis of the calculated rotational diffusion coefficients for the individual molecular axes gave a more complex picture. The diffusion was highly anisotropic and changes in temperature and solvent type led to nonuniform variation of the diffusion coefficients. The nature of these changes was rationalized based on analysis of variation of solvation patterns with temperature and solvent

Permeation of the three aromatic dipeptides through lipid bilayers: Experimental and computational study

Publisher's note added August 2016: "This article was originally published online on 27 June 2016 with a sentence missing in the Acknowledgments. After the funding acknowledgments, it should read, “G.S.J. would like to thank Wilson R. Veras Tavarez and Elizabeth De Leon Olmeda of UCC for helpful comments.” AIP Publishing apologizes for this error. All online versions of the article were corrected on 28 June 2016; the article is correct as it appears in the printed version of the journal."The time-resolved parallel artificial membrane permeability assay with fluorescence detection and comprehensive computer simulations are used to study the passive permeation of three aromatic dipeptides—N-acetyl-phenylalanineamide (NAFA), N-acetyltyrosineamide (NAYA), and N-acetyltryptophanamide (NATA) through a 1,2-dioleoyl-sn-glycero-3-phospocholine (DOPC) lipid bilayer. Measured permeation times and permeability coefficients show fastest translocation for NAFA, slowest for NAYA, and intermediate for NATA under physiological temperature and pH. Computationally, we perform umbrella sampling simulations to model the structure, dynamics, and interactions of the peptides as a function of z, the distance from lipid bilayer. The calculated profiles of the potential of mean force show two strong effects—preferential binding of each of the three peptides to the lipid interface and large free energy barriers in the membrane center. We use several approaches to calculate the position-dependent translational diffusion coefficients D(z), including one based on numerical solution the Smoluchowski equation. Surprisingly, computed D(z) values change very little with reaction coordinate and are also quite similar for the three peptides studied. In contrast, calculated values of sidechain rotational correlation times τrot(z) show extremely large changes with peptide membrane insertion—values become 100 times larger in the headgroup region and 10 times larger at interface and in membrane center, relative to solution. The peptides’ conformational freedom becomes systematically more restricted as they enter the membrane, sampling α and β and C7eq basins in solution, α and C7eq at the interface, and C7eq only in the center. Residual waters of solvation remain around the peptides even in the membrane center. Overall, our study provides an improved microscopic understanding of passive peptide permeation through membranes, especially on the sensitivity of rotational diffusion to position relative to the bilayer. Published by AIP Publishing. [http://dx.doi.org/10.1063/1.4954241

The Structure of DNA within Cationic Lipid/DNA Complexes

The structure of DNA within CLDCs used for gene delivery is controversial. Previous studies using CD have been interpreted to indicate that the DNA is converted from normal B to C form in complexes. This investigation reexamines this interpretation using CD of model complexes, FTIR as well as Raman spectroscopy and molecular dynamics simulations to address this issue. CD spectra of supercoiled plasmid DNA undergo a significant loss of rotational strength in the signal near 275 nm upon interaction with either the cationic lipid dimethyldioctadecylammonium bromide or 1,2-dioleoyltrimethylammonium propane. This loss of rotational strength is shown, however, by both FTIR and Raman spectroscopy to occur within the parameters of the B-type conformation. Contributions of absorption flattening and differential scattering to the CD spectra of complexes are unable to account for the observed spectra. Model studies of the CD of complexes prepared from synthetic oligonucleotides of varying length suggest that significant reductions in rotational strength can occur within short stretches of DNA. Furthermore, some alteration in the hydrogen bonding of bases within CLDCs is indicated in the FTIR and Raman spectroscopy results. In addition, alterations in base stacking interactions as well as hydrogen bonding are suggested by molecular dynamics simulations. A global interpretation of all of the data suggests the DNA component of CLDCs remains in a variant B form in which base/base interactions are perturbed

Unassisted Transport of N-acetyl-L-tryptophanamide through Membrane: Experiment and Simulation of Kinetics

Cellular transport machinery, such as channels and pumps, is working against the background of unassisted material transport through membranes. The permeation of a blocked tryptophan through a 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC) membrane is investigated to probe unassisted or physical transport. The transport rate is measured experimentally and modeled computationally. The time scale measured by Parallel Artificial Membrane Permeation Assay (PAMPA) experiments is ~8 h. Simulations with the Milestoning algorithm suggest Mean First Passage Time (MFPT) of ~4 h and the presence of a large barrier at the center of the bilayer. A similar calculation with the solubility-diffusion model yields MFPT of ~15 min. This permeation rate is nine orders of magnitude slower than the permeation rate of only a tryptophan side chain (computed by us and others). This difference suggests critical dependence of transport time on permeant size and hydrophilicity. Analysis of the simulation results suggests that the permeant partially preserves hydrogen bonding of the peptide backbone to water and lipid molecules even when it is moving closer to the bilayer center. As a consequence, defects of the membrane structure are developed to assist permeation