Dynamic elements and kinetics: Most favorable conformations of peptides in solution with measurements and simulations.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

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

Thermodynamically 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.

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

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-acetyl-tryptophanamide (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 ß 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.

Speed limit of protein folding evidenced in secondary structure dynamics.

As the simplest and most prevalent motif of protein folding, α-helix initiation is the starting point of macromolecular complexity. In this work, helix initiation was directly measured via ultrafast temperature-jump spectroscopy on the smallest possible helix nucleus for which only the first turn is formed. The rate's dependence on sequence, length, and temperature reveals the fastest possible events in protein folding dynamics, and it was possible to separate the rate-limiting torsional (conformational) diffusion from the fast annealing of the helix. An analytic coarse-grained model for this process, which predicts the initiation rate as a function of temperature, confirms this picture. Moreover, the stipulations of the model were verified by ensemble-converging all-atom molecular dynamics simulations, which reproduced both the picosecond annealing and the nanosecond diffusion processes observed experimentally.

GB1 hairpin kinetics: capturing the folding pathway with molecular dynamics, replica exchange and optimal dimensionality reduction.

We have performed molecular dynamics (MD) and replica-exchange (REMD) simulations of folding of the GB1 hairpin peptide in aqueous solution. REMD results were consistent with a cooperative zipper folding model. 120 µs MD trajectories at 320 K yielded relaxation times of 1.8 µs and 100 ns, with the slower assigned to global folding. The MD folding/unfolding transitions also followed the cooperative zipper model, specifying nucleation at the central turn followed by consecutive hydrogen bond formation. Formation of hydrogen bonds and hydrophobic contacts were highly correlated. Coarse-grained kinetic models constructed with the Optimal Dimensionality Reduction (ODR) approach found a folding time of 3.3 µs and unfolding time of 4.0 µs. Additionally, relaxation times in the 130-170 ns range could be assigned to formation of the transition state and off-path intermediates. The unfolded state was the most highly populated and, significantly, most heterogenous, assembling the largest number of microstates, primarily composed of extended and turn structures. The folded state was also heterogenous, but a to a lesser degree, involving the fully folded and partially folded in-register hairpins at early stages of the zipper pathway. The transition state corresponded to the nucleated hairpin, with central turn and first beta-sheet hydrogen bond, while the off-path intermediates were off-register partial hairpins. Our simulation results were in excellent agreement with experimental data on folded fraction, relaxation time and folding mechanism. The new findings from this work suggest a highly cooperative zipper folding mechanism, nascent hairpin transition state and ∼100 ns relaxation related to intermediate formation.Communicated by Ramaswamy H. Sarma.

Observing reorientation dynamics with Time-Resolved fluorescence and molecular dynamics in varying periodic boundary conditions.

This work presents a combined study of time-resolved fluorescence spectroscopy and all-atom molecular dynamics simulation to investigate periodic boundary conditions' and water models' influence on the orientation dynamics and translational and rotational diffusion of peptides in solution. We have characterized the effects of solvent box size and water model choice on the dynamics of two peptide systems, NATA and WK5. Computationally, translational, and rotational diffusion and internal fluctuations are investigated through all-atom molecular dynamics simulation with two water models and different box sizes. These results are compared with time-resolved fluorescence anisotropy decay (FAD) measurements. The associated time constant and orientation dynamics from FAD measurement along the 1Lb axis provided baseline data to validate molecular dynamics simulation. The modeling results show that diffusion rates vary roughly in inverse proportion to water model viscosity, as one would expect. Corrections for finite box size are significant for translational diffusion and insignificant for rotational diffusion. This study also finds that internal dynamics described by autocorrelation functions and kinetic network models are relatively insensitive to both box size and water model properties. Our observation suggests that different peptide properties respond differently to a change in simulation conditions.Communicated by Ramaswamy H. Sarma.

Unassisted N-acetyl-phenylalanine-amide transport across membrane with varying lipid size and composition: kinetic measurements and atomistic molecular dynamics simulation.

Biological membranes are essential to preserve structural integrity and regulate functional properties through the permeability of nutrients, pharmaceutical drugs, and neurotransmitters of a living cell. The movement of acetylated and amidated phenylalanine (NAFA) across 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane bilayers is investigated to probe physical transport. The rate of transport is measured experimentally applying parallel artificial membrane permeation assay (PAMPA). At the physiological temperature, 310 K, the measured time constants in the neutral pH were ∼6 h in DOPC and ∼3 h in POPC, while in a more acidic condition, at a pH 4.8, the time constants were ∼8 h in both lipids. Computationally, we have expanded our transport study of three aromatic dipeptides across a bilayer composed of DOPC18. In this study, we have examined the effects of lipid composition and bilayer size on the passive transport of NAFA by simulating the dipeptide in three different bilayers, with 50 DOPC lipids, 50 POPC lipids, and 40 POPC molecules. Specifically, atomistic molecular dynamics simulations with umbrella sampling were used to calculate the potential of mean force for the passive permeation of NAFA across the bilayers. Diffusion constants were then calculated by numerically solving the Smoluchowski equation. Permeability coefficients and mean first passage times were then calculated. Structural properties - Ramachandran plots, sidechain torsions, peptide insertion angles, radial distribution functions, and proximal peptide water molecules - were also examined to determine the effect of system size and lipid type. In terms of systems size, we observed a small decrease in the highest barrier of the potential of mean force and fewer sampled sidechain dihedral angle conformations with 40 versus 50 POPC lipids due to weaker membrane deformations within a smaller lipid bilayer. In terms of lipid type, DOPC contains two monounsaturated acyl chains compared to only one such acyl chain in POPC; therefore, DOPC bilayers are less ordered and more easily deformed, as seen by a much broader potential of mean force profile. The NAFA in DOPC lipid also transitioned to an internally hydrogen-bonded backbone conformation at lower membrane depths than in POPC. Similarly, as for other aromatic dipeptides, NAFA tends to insert into the membrane sidechain-first, remains mostly desolvated in the membrane center, and exhibits slow reorientations within the bilayer in both DOPC and POPC. With a joint experimental and computational study we have gained a new insight into the rate of transport and the underlying microscopic mechanism in different lipid bilayer conditions of the simplest hydrophobic aromatic dipeptide.Communicated by Ramaswamy H. Sarma.

Probing the Internal Dynamics and Shape of Simple Peptides in Urea, Guanidinium Hydrochloride, and Proline Solutions with Time-Resolved Fluorescence Anisotropy and Atomistic Cosolvent Simulations.

Picosecond time-resolved fluorescence anisotropy was used to measure the effect of denaturants and osmolytes on the reorientation dynamics of the simplest dipeptide. The solvent denaturants guanidinium hydrochloride (gdm), urea, and the osmolyte proline were used at several concentrations. Analysis of the concentration dependence of denaturants at a fixed temperature showed faster and slower reorientation time in two different denaturants at a nearly identical solvent viscosity (η). The reorientation time τ significantly deviates from Kramers' theory (τ ∝ η1) in the high friction limit for guanidinium and urea with r ≈ 0.4 and r ≈ 0.6 at pH 7.2, respectively. In proline, τ is nearly proportional to η. Atomistic molecular dynamics simulations of the dipeptide in identical cosolvents showed excellent agreement with the measured rotational orientation time. The dipeptide dihedral (ϕ, ψ) isomerization times in water and 6 M urea are almost identical and significantly slower in guanidinium. If a faster and slower reorientation time can be associated with the compact and expanded shapes, the fractional viscosity dependence for guanidinium and urea may result from the fact that internal dynamics of peptides in these cosolvents involve higher and lower internal friction within the dynamic elements.

Dissecting Multiple Pathways in the Relaxation Dynamics of Helix <==> Coil Transitions with Optimum Dimensionality Reduction.

Fast kinetic experiments with dramatically improved time resolution have contributed significantly to understanding the fundamental processes in protein folding pathways involving the formation of a-helices and b-hairpin, contact formation, and overall collapse of the peptide chain. Interpretation of experimental results through application of a simple statistical mechanical model was key to this understanding. Atomistic description of all events observed in the experimental findings was challenging. Recent advancements in theory, more sophisticated algorithms, and a true long-term trajectory made way for an atomically detailed description of kinetics, examining folding pathways, validating experimental results, and reporting new findings for a wide range of molecular processes in biophysical chemistry. This review describes how optimum dimensionality reduction theory can construct a simplified coarse-grained model with low dimensionality involving a kinetic matrix that captures novel insights into folding pathways. A set of metastable states derived from molecular dynamics analysis generate an optimally reduced dimensionality rate matrix following transition pathway analysis. Analysis of the actual long-term simulation trajectory extracts a relaxation time directly comparable to the experimental results and confirms the validity of the combined approach. The application of the theory is discussed and illustrated using several examples of helix <==> coil transition pathways. This paper focuses primarily on a combined approach of time-resolved experiments and long-term molecular dynamics simulation from our ongoing work.

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.

Synthesis and evaluation of a near-infrared fluorescent non-peptidic bivalent integrin alpha(v)beta(3) antagonist for cancer imaging.

Computer modeling approaches to identify new inhibitors are essentially a very sophisticated and efficient way to design drugs. In this study, a bivalent nonpeptide intergrin alpha(v)beta(3) antagonist (bivalent IA) has been synthesized on the basis of an in silico rational design approach. A near-infrared (NIR) fluorescent imaging probe has been developed from this bivalent compound. In vitro binding assays have shown that the bivalent IA (IC(50) = 0.40 +/- 0.11 nM) exhibited improved integrin alpha(v)beta(3) affinity in comparison with the monovalent IA (IC(50) = 22.33 +/- 4.51 nM), resulting in an over 50-fold improvement in receptor affinity. NIR imaging probe, bivalent-IA-Cy5.5 conjugate, also demonstrated significantly increased binding affinity (IC(50) = 0.13 +/- 0.02 nM). Fluorescence microscopy studies showed integrin-mediated endocytosis of bivalent-IA-Cy5.5 in U87 cells which was effectively blocked by nonfluorescent bivalent IA. We also demonstrated tumor accumulation of this NIR imaging probe in U87 mouse xenografts.

Synthesis and evaluation of bivalent, peptidomimetic antagonists of the αvß3 integrins.

Targeting the integrin α(v)ß(3) by directly interfering with its function is considered to be an effective and non-cytotoxic strategy for the treatment of tumor. In this study, a series of bivalent analogs of peptidomimetic integrin antagonists IA 1 and IAC 2 were designed, synthesized, and evaluated for their ability to inhibit the integrin α(v)ß(3). All the bivalent ligands exhibited increased potency compared to that of their monomeric counterparts for the integrin α(v)ß(3) with low nanomolar range binding affinity. The best bivalent ligand 6 tested in the series has an IC(50)=0.09 nM evaluated by ELISA assay. We conclude that multivalency is providing a useful template for the development novel integrin α(v)ß(3) antagonists as potential therapeutics.

Reorientations of aromatic amino acids and their side chain models: anisotropy measurements and molecular dynamics simulations.

We present a study of reorientation dynamics of the three aromatic amino acids and their side chain models in aqueous solution. Experimentally, anisotropy decay measurements with picosecond time resolution were performed for blocked tryptophan, tyrosine, and phenyalanine and model compounds p-cresol and 3-methylindole. Computationally, rotational diffusion was modeled by molecular dynamics simulations for the three aromatic residues and their side chain models: benzene, toluene, phenol, p-cresol, indole, and 3-methylindole in explicit water. Our simulations used the CHARMM protein force field and associated TIP3P water model and tend to underestimate the rotational correlation times. However, the simulations yield several interesting qualitative insights into reorientational motions that complement the experimental measurements. The effects of substituent and temperature on reorientations of the parent compounds are well reproduced computationally. Additionally, simulations indicate strongly anisotropic reorientations for most of the studied compounds and a separation of time scales between conformational dynamics and rotational diffusion. Comparison with continuum hydrodynamic models suggests that we may consider that the blocked amino acids move under stick boundary conditions, while the dynamics for most of the model compounds falls between stick and slip conditions. Our systematic treatment of blocked amino acids, starting from the parent compounds (benzene, phenol, and indole) provides a baseline for understanding the anisotropy decay signals of more complicated peptide systems.

Helix formation in a pentapeptide: experiment and force-field dependent dynamics.

We used a combined approach of experiment and simulation to determine the helical population and folding pathway of a small helix forming blocked pentapeptide, Ac-(Ala)(5)-NH(2). Experimental structural characterization of this blocked peptide was carried out with far UV circular dichroism spectroscopy, FTIR, and NMR measurements. These measurements confirm the presence of the α-helical state in a buffer solution. Direct molecular dynamics and replica-exchange simulations of the pentapeptide were performed using several popular force fields with explicit solvent. The simulations yielded statistically reliable estimates of helix populations, melting curves, folding, and nucleation times. The distributions of conformer populations are used to measure folding cooperativity. Finally, a statistical analysis of the sample of helix-coil transition paths was performed. The details of the calculated helix populations, folding kinetics and pathways vary with the employed force field. Interestingly, the helix populations, folding, and unfolding times obtained from most of the studied force fields are in qualitative agreement with each other and with available experimental data, with the deviations corresponding to several kcal/mol in energy at 300 K. Most of the force fields also predict qualitatively similar transition paths, with unfolding initiated at the C-terminus. Accuracy of potential energy parameters, rather than conformational sampling may be the limiting factor in current molecular simulations.

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 kinetics 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 psi dihedral angle) that are assisted by significant nonmonotonic fluctuations of nearby torsions. The most effective unfolding pathway is via the N-terminal.

Deprotonation of a Single Amino Acid Residue Induces Significant Stability in an α-Helical Heteropeptide.

Stability of secondary structural elements is an integral component of a structurally stable protein. Presence of protons in the residue sequence and their immediate environment play a significant role in conformational stability. In this study, we show that removing a proton from a single amino acid residue significantly increases the stability of an α-helical heteropeptide in comparison with the unprotonated form. Far-UV circular dichroism spectroscopy, fluorescence spectroscopy, fluorescence energy transfer measurements, and over 10 µs of all-atom molecular dynamics simulations are used to provide an atomically detailed characterization of this event. There is a single histidine residue in the studied α-helical peptide sequence toward the N-terminal that interacts with a tryptophan located four residues away and quenches the fluorescence when protonated. Removing a proton from this histidine residue dequenches the tryptophan fluorescence and contributes to a significant increase in the helix stability. Atomically detailed analysis of individual residue conformations shows that the protonated histidine tends to be in closer proximity to the tryptophan, which correlates with higher helix content in the N and C termini and lower helix content in the central region of the peptide. In the presence of a neutral histidine, when tryptophan fluorescence is no longer quenched and histidine moves further away from tryptophan, the helix content remains mostly unchanged in the N-and-C termini and significantly increases in the central region. Our results strongly suggest that interactions of the tryptophan with a protonated histidine downregulate the helix population in the central segment of the helical structure compared to a neutral histidine residue. Upregulation of helix population of the central segment of this α-helical heteropeptide in the presence of a neutral histidine residue significantly increases the peptide structural stability.

Helix-Coil Transition Courses Through Multiple Pathways and Intermediates: Fast Kinetic Measurements and Dimensionality Reduction.

Nanosecond laser temperature jumps with tryptophan fluorescence detection and molecular dynamics simulation with kinetic dimensionality reduction were used to study the helix-coil transition in a 21-residue α-helical heteropeptide. Analysis of the temperature- dependent relaxation dynamics of this heteropeptide identified a distinct faster component of 20-35 ns, besides a slower component of 300-400 ns at temperatures between 296 and 280 K. To understand the mechanism of progression from a non-structured coil state to a structured helical state, we carried out a 12 µs molecular dynamics simulation of this peptide system. Clustering and optimal dimensionality reduction were applied to the molecular dynamics trajectory to generate low-dimensional coarse-grained models of the underlying kinetic network in terms of 2-5 metastable states. In accord with the generally accepted understanding of the multiple conformations and high entropy of the unfolded ensemble of states, the "coil" metastable set contains the largest number of structures. Interestingly, the helix metastable state was also found to be structurally heterogeneous, consisting of the completely helical form and several partly folded conformers that interconvert at a time scale faster that global folding. The intermediate states contain the fewest structures, have lowest populations, and have the shortest lifetimes. As the number of considered metastable states increases, more intermediates and more folding paths appear in the coarse-grained models. One of these intermediates corresponds to the transition state for folding, which involves an "off-center" helical region over residues 11-16. The kinetic network model is consistent with a statistical picture of folding following a simple reaction coordinate counting the helical population of individual residues. On the basis of simulations, we propose that the fast relaxation time should be assigned to cooperative folding/unfolding of segments of 1-4 neighboring residues.

Probing Selection Mechanism of the Most Favorable Conformation of a Dipeptide in Chaotropic and Kosmotropic Solution.

Chaotropes like urea and guanidinium chloride (GdmCl) tend to destabilize, and kosmotropes like proline tend to stabilize folded structures of peptides and proteins. Here, we combine fluorescence anisotropy decay measurements and molecular dynamics simulations to gain a microscopic understanding of the molecular mechanism for shifting conformational preferences in aqueous, GdmCl, urea, and proline solutions of a simple model dipeptide, N-acetyl-tryptophan-amide (NATA). Measured anisotropy decay of NATA as a function of temperature, pH, and cosolvent concentrations showed reorientations moderately slower in GdmCl and urea and substantially slower in proline compared to those of aqueous environment. A small change in pH significantly slows orientation time in water and GdmCl and less markedly in urea. Computationally, we use molecular dynamics with dihedral restraints to separately analyze the motions and interactions of the representative NATA conformers in the four different solvent environments. This novel analysis provides a dissection of the observed overall diffusion rates into contributions from individual dipeptide conformations. The variation of rotational diffusion rates with conformation are quite large. Population-weighted averaging or using properties of the major cluster reproduces the dynamical features of the full unrestrained dynamics. Additionally, we correlate the observable diffusion rates with microscopic features of conformer size, shape, and solvation. This analysis uncovered underlying differences in detailed atomistic behavior of the three cosolvents-urea, GdmCl, and proline. For both urea and the pure water system we find good agreement with hydrodynamic theory, with diffusion rates primarily correlated with conformer size and shape. In contrast, for GdmCl and proline solutions, the variation in conformer diffusion rates was mostly determined by specific interactions with the cosolvents. We also find preferences for different molecular shapes by the three cosolvents, with increased preferential solvation of smaller and more spherical conformers by urea and larger and more elongated conformers by GdmCl and proline. Additionally, our results provide a basis for a simple approximate model of the effects of pH lowering on dipeptide conformational equilibria. The translational diffusion rates of NATA are less sensitive to conformations, but variation with solvation strength is similar to rotational diffusion. Our results, combining experiment and simulation, show that we can identify the individual peptide conformers with definite microscopic properties of shape, size, and solvation, that are responsible for producing physical observables, such as translational and orientational diffusion in the complex solvent environments of denaturants and osmolytes.

Reorientation Motion and Preferential Interactions of a Peptide in Denaturants and Osmolyte.

Fluorescence anisotropy decay measurements and all atom molecular dynamics simulations are used to characterize the orientational motion and preferential interaction of a peptide, N-acetyl-tryptophan-amide (NATA) containing two peptide bonds, in aqueous, urea, guanidinium chloride (GdmCl), and proline solution. Anisotropy decay measurements as a function of temperature and concentration showed moderate slowing of reorientations in urea and GdmCl and very strong slowing in proline solution, relative to water. These effects deviate significantly from simple proportionality of peptide tumbling time to solvent viscosity, leading to the investigation of microscopic preferential interaction behavior through molecular dynamics simulations. Examination of the interactions of denaturants and osmolyte with the peptide backbone uncovers the presence of strongest interaction with urea, intermediate with proline, and weakest with GdmCl. In contrast, the strongest preferential solvation of the peptide side chain is by the nonpolar part of the proline zwitterion, followed by urea, and GdmCl. Interestingly, the local density of urea around the side chain is higher, but the GdmCl distribution is more organized. Thus, the computed preferential solvation of the side chain by the denaturants and osmolyte can account for the trend in reorientation rates. Analysis of water structure and its dynamics uncovered underlying differences between urea, GdmCl, and proline. Urea exerted the smallest perturbation of water behavior. GdmCl had a larger effect on water, slowing kinetics and stabilizing interactions. Proline had the largest overall interactions, exhibiting a strong stabilizing effect on both water-water and water-peptide hydrogen bonds. The results for this elementary peptide system demonstrate significant differences in microscopic behavior of the examined solvent environments. For the commonly used denaturants, urea tends to form disorganized local aggregates around the peptide groups and has little influence on water, while GdmCl only forms specific interactions with the side chain and tends to destabilize water structure. The protective osmolyte proline has the strongest and most specific interactions with the tryptophan side chain, and also stabilizes both water-water and water-peptide hydrogen bonds. Our results strongly suggest protein or peptide denaturation triggered by urea occurs by direct interaction, whereas GdmCl interacts favorably with side chains and destabilizes peptide-water hydrogen bonds. The stabilization of biopolymers by an osmolyte such as proline is governed by favorable preferential interaction with the side chains and stabilization of water.