Foldamers controlled by functional triamino acids: structural investigation of α/γ-hybrid oligopeptides.

Peptide-like foldamers controlled by normal amide backbone hydrogen bonding have been extensively studied, and their folding patterns largely rely on configurational and conformational constraints induced by the steric properties of backbone substituents at appropriate positions. In contrast, opportunities to influence peptide secondary structure by functional groups forming individual hydrogen bond networks have not received much attention. Here, peptide-like foldamers consisting of alternating α,ß,γ-triamino acids 3-amino-4-(aminomethyl)-2-methylpyrrolidine-3-carboxylate (AAMP) and natural amino acids glycine and alanine are reported, which were obtained by solution phase peptide synthesis. They form ordered secondary structures, which are dominated by a three-dimensional bridged triazaspiranoid-like hydrogen bond network involving the non-backbone amino groups, the backbone amide hydrogen bonds, and the relative configuration of the α,ß,γ-triamino and α-amino acid building blocks. This additional stabilization leads to folding in both nonpolar organic as well as in aqueous environments. The three-dimensional arrangement of the individual foldamers is supported by X-ray crystallography, NMR spectroscopy, chiroptical methods, and molecular dynamics simulations.

Building Water Models Compatible with Charge Scaling Molecular Dynamics.

Charge scaling has proven to be an efficient way to account in a mean-field manner for electronic polarization by aqueous ions in force field molecular dynamics simulations. However, commonly used water models with dielectric constants over 50 are not consistent with this approach leading to "overscaling", i.e., generally too weak ion-ion interactions. Here, we build water models fully compatible with charge scaling, i.e., having the correct low-frequency dielectric constant of about 45. To this end, we employ advanced optimization and machine learning schemes in order to explore the vast parameter space of four-site water models efficiently. As an a priori unwarranted positive result, we find a sizable range of force field parameters that satisfy the above dielectric constant constraint providing at the same time accuracy with respect to experimental data comparable with the best existing four-site water models such as TIP4P/2005, TIP4P-FB, or OPC. The present results thus open the way to the development of a consistent charge scaling force field for modeling ions in aqueous solutions.

Hydration of biologically relevant tetramethylammonium cation by neutron scattering and molecular dynamics.

Neutron scattering and molecular dynamics studies were performed on a concentrated aqueous tetramethylammonium (TMA) chloride solution to gain insight into the hydration shell structure of TMA, which is relevant for understanding its behavior in biological contexts of, e.g., properties of phospholipid membrane headgroups or interactions between DNA and histones. Specifically, neutron diffraction with isotopic substitution experiments were performed on TMA and water hydrogens to extract the specific correlation between hydrogens in TMA (HTMA) and hydrogens in water (HW). Classical molecular dynamics simulations were performed to help interpret the experimental neutron scattering data. Comparison of the hydration structure and simulated neutron signals obtained with various force field flavors (e.g. overall charge, charge distribution, polarity of the CH bonds and geometry) allowed us to gain insight into how sensitive the TMA hydration structure is to such changes and how much the neutron signal can capture them. We show that certain aspects of the hydration, such as the correlation of the hydrogen on TMA to hydrogen on water, showed little dependence on the force field. In contrast, other correlations, such as the ion-ion interactions, showed more marked changes. Strikingly, the neutron scattering signal cannot discriminate between different hydration patterns. Finally, ab initio molecular dynamics was used to examine the three-dimensional hydration structure and thus to benchmark force field simulations. Overall, while neutron scattering has been previously successfully used to improve force fields, in the particular case of TMA we show that it has only limited value to fully determine the hydration structure, with other techniques such as ab initio MD being of a significant help.

Nonaqueous Ion Pairing Exemplifies the Case for Including Electronic Polarization in Molecular Dynamics Simulations.

The inclusion of electronic polarization is of crucial importance in molecular simulations of systems containing charged moieties. When neglected, as often done in force field simulations, charge-charge interactions in solution may become severely overestimated, leading to unrealistically strong bindings of ions to biomolecules. The electronic continuum correction introduces electronic polarization in a mean-field way via scaling of charges by the reciprocal of the square root of the high-frequency dielectric constant of the solvent environment. Here, we use ab initio molecular dynamics simulations to quantify the effect of electronic polarization on pairs of like-charged ions in a model nonaqueous environment where electronic polarization is the only dielectric response. Our findings confirm the conceptual validity of this approach, underlining its applicability to complex aqueous biomolecular systems. Simultaneously, the results presented here justify the potential employment of weaker charge scaling factors in force field development.

Dynamics of transition dipole moment orientation in representative fluorescent proteins.

Molecules of fluorescent proteins (FPs) exhibit distinct optical directionality. This optical directionality is characterized by transition dipole moments (TDMs), and their orientation with respect to the molecular structures. Although our recent observations of FP crystals allowed us to determine the mean TDM directions with respect to the framework of representative FP molecules, the dynamics of TDM orientations within FP molecules remain to be ascertained. Here we describe the results of our investigations of the dynamics of TDM directions in the fluorescent proteins eGFP, mTurquoise2 and mCherry, through time-resolved fluorescence polarization measurements and microsecond time scale all-atom molecular dynamics (MD) simulations. The investigated FPs exhibit initial fluorescence anisotropies (r0) consistent with significant differences in the orientation of the excitation and emission TDMs. However, based on MD data, we largely attribute this observation to rapid (sub-nanosecond) fluorophore motions within the FP molecular framework. Our results allow improved determinations of orientational distributions of FP molecules by polarization microscopy, as well as more accurate interpretations of fluorescence resonance energy transfer (FRET) observations.

Common Cations Are Not Polarizable: Effects of Dispersion Correction on Hydration Structures from Ab Initio Molecular Dynamics.

We employed density functional theory-based ab initio molecular dynamics simulations to examine the hydration structure of several common alkali and alkali earth metal cations. We found that the commonly used atom pairwise dispersion correction scheme D3, which assigns dispersion coefficients based on the neutral form of the atom rather than its actual oxidation state, leads to inaccuracies in the hydration structures of these cations. We evaluated this effect for lithium, sodium, potassium, and calcium and found that the inaccuracies are particularly pronounced for sodium and potassium compared to the experiment. To remedy this issue, we propose disabling the D3 correction specifically for all cation-including pairs, which leads to a much better agreement with experimental data.

Curvature Matters: Modeling Calcium Binding to Neutral and Anionic Phospholipid Bilayers.

In this work, the influence of membrane curvature on the Ca2+ binding to phospholipid bilayers is investigated by means of molecular dynamics simulations. In particular, we compared Ca2+ binding to flat, elastically buckled, or uniformly bent zwitterionic and anionic phospholipid bilayers. We demonstrate that Ca2+ ions bind preferably to the concave membrane surfaces in both types of bilayers. We also show that the membrane curvature leads to pronounced changes in Ca2+ binding including differences in free ion concentrations, lipid coordination distributions, and the patterns of ion binding to different chemical groups of lipids. Moreover, these effects differ substantially for the concave and convex membrane monolayers. Comparison between force fields with either full or scaled charges indicates that charge scaling results in reduction of the Ca2+ binding to curved phosphatidylserine bilayers, while for phosphatidylcholine membranes, calcium binds only weakly for both force fields.

Influence of electronic polarization on the binding of anions to a chloride-pumping rhodopsin.

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

Ionic Strength and Solution Composition Dictate the Adsorption of Cell-Penetrating Peptides onto Phosphatidylcholine Membranes.

Adsorption of arginine-rich positively charged peptides onto neutral zwitterionic phosphocholine (PC) bilayers is a key step in the translocation of those potent cell-penetrating peptides into the cell interior. In the past, we have shown both theoretically and experimentally that polyarginines adsorb to the neutral PC-supported lipid bilayers in contrast to polylysines. However, comparing our results with previous studies showed that the results often do not match even at the qualitative level. The adsorption of arginine-rich peptides onto 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) may qualitatively depend on the actual experimental conditions where binding experiments have been performed. In this work, we systematically studied the adsorption of R9 and K9 peptides onto the POPC bilayer, aided by molecular dynamics (MD) simulations and fluorescence cross-correlation spectroscopy (FCCS) experiments. Using MD simulations, we tested a series of increasing peptide concentrations, in parallel with increasing Na+ and Ca2+ salt concentrations, showing that the apparent strength of adsorption of R9 decreases upon the increase of peptide or salt concentration in the system. The key result from the simulations is that the salt concentrations used experimentally can alter the picture of peptide adsorption qualitatively. Using FCCS experiments with fluorescently labeled R9 and K9, we first demonstrated that the binding of R9 to POPC is tighter by almost 2 orders of magnitude compared to that of K9. Finally, upon the addition of an excess of either Na+ or Ca2+ ions with R9, the total fluorescence correlation signal is lost, which implies the unbinding of R9 from the PC bilayer, in agreement with our predictions from MD simulations.

Efficiently Computing NMR 1H and 13C Chemical Shifts of Saccharides in Aqueous Environment.

Determining the structure of saccharides in their native environment is crucial to understanding their function and more accurately targeting their utilization. Nuclear magnetic resonance observables such as the nuclear Overhauser effect or spin-spin coupling constants are routinely utilized to study saccharides in their native water environment. However, while highly sensitive to the local environment, chemical shifts are mostly overlooked, despite being commonly measured for compounds identification. Although chemical shifts carry considerable structural information, their direct association with structure is notoriously difficult. This is mostly due to the similarity in the chemical nature of most saccharides causing similar physicochemical environments close to sugar C and H atoms, resulting in comparable chemical shifts. The rise of computational power allows one to compute reliable chemical shifts and use them to determine atomistic details of these sugars in solution. However, any prediction is severely limited by the computational protocol used and its accuracy. In this work, we studied a set of 31 saccharides on which we evaluated various computational protocols to calculate the total number of 375 1H and 327 13C chemical shifts of sugars in an aqueous environment. Our study proposes two cost-effective protocols for simulating 1H and 13C chemical shifts that we recommend for further use. These protocols can help with the interpretation of experimental spectra, but we also show that they are also capable of structure prediction independently. This is possible because of the low mean absolute deviations of calculated shifts from the experiment (0.06 ppm for 1H and 1.09 ppm for 13C). We explore different solvation methods, basis sets, and optimization schemes to reach such accuracy. A correct sampling of the conformation phase space of flexible sugar molecules is also key to obtaining accurately converged theoretical chemical shifts. The linear regression method was applied to convert the calculated isotropic nuclear magnetic shielding constants to simulated chemical shifts comparable with the experiment. The achieved level of accuracy can help in utilizing chemical shifts for elucidating the 3D atomistic structure of saccharides in aqueous solutions. All linear regression parameters obtained on our extensive set of sugars for all the tested protocols can be reutilized in future works.

Changes in the Local Conformational States Caused by Simple Na+ and K+ Ions in Polyelectrolyte Simulations: Comparison of Seven Force Fields with and without NBFIX and ECC Corrections.

Electrostatic interactions have a determining role in the conformational and dynamic behavior of polyelectrolyte molecules. In this study, anionic polyelectrolyte molecules, poly(glutamic acid) (PGA) and poly(aspartic acid) (PASA), in a water solution with the most commonly used K+ or Na+ counterions, were investigated using atomistic molecular dynamics (MD) simulations. We performed a comparison of seven popular force fields, namely AMBER99SB-ILDN, AMBER14SB, AMBER-FB15, CHARMM22*, CHARMM27, CHARMM36m and OPLS-AA/L, both with their native parameters and using two common corrections for overbinding of ions, the non-bonded fix (NBFIX), and electronic continuum corrections (ECC). These corrections were originally introduced to correct for the often-reported problem concerning the overbinding of ions to the charged groups of polyelectrolytes. In this work, a comparison of the simulation results with existing experimental data revealed several differences between the investigated force fields. The data from these simulations and comparisons with previous experimental data were then used to determine the limitations and strengths of these force fields in the context of the structural and dynamic properties of anionic polyamino acids. Physical properties, such as molecular sizes, local structure, and dynamics, were studied using two types of common counterions, namely potassium and sodium. The results show that, in some cases, both the macroion size and dynamics depend strongly on the models (parameters) for the counterions due to strong overbinding of the ions and charged side chain groups. The local structures and dynamics are more sensitive to dihedral angle parameterization, resulting in a preference for defined monomer conformations and the type of correction used. We also provide recommendations based on the results.

Use of Raman and Raman optical activity to extract atomistic details of saccharides in aqueous solution.

Sugars are crucial components in biosystems and industrial applications. In aqueous environments, the natural state of short saccharides or charged glycosaminoglycans is floating and wiggling in solution. Therefore, tools to characterize their structure in a native aqueous environment are crucial but not always available. Here, we show that a combination of Raman/ROA and, on occasions, NMR experiments with Molecular Dynamics (MD) and Quantum Mechanics (QM) is a viable method to gain insights into structural features of sugars in solutions. Combining these methods provides information about accessible ring puckering conformers and their proportions. It also provides information about the conformation of the linkage between the sugar monomers, i.e., glycosidic bonds, allowing for identifying significantly accessible conformers and their relative abundance. For mixtures of sugar moieties, this method enables the deconvolution of the Raman/ROA spectra to find the actual amounts of its molecular constituents, serving as an effective analytical technique. For example, it allows calculating anomeric ratios for reducing sugars and analyzing more complex sugar mixtures to elucidate their real content. Altogether, we show that combining Raman/ROA spectroscopies with simulations is a versatile method applicable to saccharides. It allows for accessing many features with precision comparable to other methods routinely used for this task, making it a viable alternative. Furthermore, we prove that the proposed technique can scale up by studying the complicated raffinose trisaccharide, and therefore, we expect its wide adoption to characterize sugar structural features in solution.

Nonconverged Constraints Cause Artificial Temperature Gradients in Lipid Bilayer Simulations.

Molecular dynamics (MD) simulations have become an indispensable tool to investigate phase separation in model membrane systems. In particular, simulations based on coarse-grained (CG) models have found widespread use due to their increased computational efficiency, allowing for simulations of multicomponent lipid bilayers undergoing phase separation into liquid-ordered and liquid-disordered domains. Here, we show that a significant temperature difference between molecule types can artificially arise in CG MD membrane simulations with the standard Martini simulation parameters in GROMACS. In particular, the linear constraint solver (LINCS) algorithm does not converge with its default settings, resulting in serious temperature differences between molecules in a time step-dependent manner. We demonstrate that the underlying reason for this behavior is the presence of highly constrained moieties, such as cholesterol. Their presence can critically impact numerous structural and dynamic membrane properties obtained from such simulations. Furthermore, any preference of these molecules toward a certain membrane phase can lead to spatial temperature gradients, which can amplify the degree of phase separation or even induce it in compositions that would otherwise mix well. We systematically investigated the effect of the integration time step and LINCS settings on membrane properties. Our data show that for cholesterol-containing membranes, a time step of 20 fs should be combined with at least lincs_iter = 2 and lincs_order = 12, while using a time step of 30 fs requires at least lincs_iter = 3 and lincs_order = 12 to bring the temperature differences to a level where they do not perturb central membrane properties. Moreover, we show that in cases where stricter LINCS settings are computationally too demanding, coupling the lipids in multiple groups to the temperature bath offers a practical workaround to the problem, although the validity of this approach should be further verified. Finally, we show that similar temperature gradients can also emerge in atomistic simulations using the CHARMM force field in combination with settings that allow for a 5 fs integration step.

Anisotropic diffusion of membrane proteins at experimental timescales.

Single-particle tracking (SPT) experiments of lipids and membrane proteins provide a wealth of information about the properties of biomembranes. Careful analysis of SPT trajectories can reveal deviations from ideal Brownian behavior. Among others, this includes confinement effects and anomalous diffusion, which are manifestations of both the nanoscale structure of the underlying membrane and the structure of the diffuser. With the rapid increase in temporal and spatial resolution of experimental methods, a new aspect of the motion of the particle, namely, anisotropic diffusion, might become relevant. This aspect that so far received only little attention is the anisotropy of the diffusive motion and may soon provide an additional proxy to the structure and topology of biomembranes. Unfortunately, the theoretical framework for detecting and interpreting anisotropy effects is currently scattered and incomplete. Here, we provide a computational method to evaluate the degree of anisotropy directly from molecular dynamics simulations and also point out a way to compare the obtained results with those available from SPT experiments. In order to probe the effects of anisotropic diffusion, we performed coarse-grained molecular dynamics simulations of peripheral and integral membrane proteins in flat and curved bilayers. In agreement with the theoretical basis, our computational results indicate that anisotropy can persist up to the rotational relaxation time [τ=(2Dr)-1], after which isotropic diffusion is observed. Moreover, the underlying topology of the membrane bilayer can couple with the geometry of the particle, thus extending the spatiotemporal domain over which this type of motion can be detected.

N-Glycosylation can selectively block or foster different receptor-ligand binding modes.

While DNA encodes protein structure, glycans provide a complementary layer of information to protein function. As a prime example of the significance of glycans, the ability of the cell surface receptor CD44 to bind its ligand, hyaluronan, is modulated by N-glycosylation. However, the details of this modulation remain unclear. Based on atomistic simulations and NMR, we provide evidence that CD44 has multiple distinct binding sites for hyaluronan, and that N-glycosylation modulates their respective roles. We find that non-glycosylated CD44 favors the canonical sub-micromolar binding site, while glycosylated CD44 binds hyaluronan with an entirely different micromolar binding site. Our findings show (for the first time) how glycosylation can alter receptor affinity by shielding specific regions of the host protein, thereby promoting weaker binding modes. The mechanism revealed in this work emphasizes the importance of glycosylation in protein function and poses a challenge for protein structure determination where glycosylation is usually neglected.

Martini 3: a general purpose force field for coarse-grained molecular dynamics.

The coarse-grained Martini force field is widely used in biomolecular simulations. Here we present the refined model, Martini 3 ( http://cgmartini.nl ), with an improved interaction balance, new bead types and expanded ability to include specific interactions representing, for example, hydrogen bonding and electronic polarizability. The updated model allows more accurate predictions of molecular packing and interactions in general, which is exemplified with a vast and diverse set of applications, ranging from oil/water partitioning and miscibility data to complex molecular systems, involving protein-protein and protein-lipid interactions and material science applications as ionic liquids and aedamers.

Resolving the Equal Number Density Puzzle: Molecular Picture from Simulations of LiCl(aq) and NaCl(aq).

The change in number densities of aqueous solutions of alkali chlorides should be qualitatively predictable. Typically, as cations get larger, the number density of the solution decreases. However, aqueous solutions of lithium and sodium chloride exhibit at ambient conditions practically identical number densities at equal molalities despite different ionic sizes. Here, we provide an atomistic interpretation of this experimentally observed anomalous behavior using molecular dynamics simulations. The obtained results show that the rigidity of the Li+ first and second solvation shells and the associated compromised hydrogen bonding result in practically equal average water densities in the local hydration regions for Li+ and Na+ despite different sizes of the cations. In addition, in more distant regions from the cations, the water densities of these two solutions also coincide. These findings thus provide an atomistic interpretation for matching number densities of LiCl and NaCl solutions. In contrast, the number density differences between NaCl and KCl solutions as well as between LiCl and KCl solutions behave in a regular fashion with lower number densities of solutions observed for larger cations.

Binding of divalent cations to acetate: molecular simulations guided by Raman spectroscopy.

In spite of the biological importance of the binding of Zn2+, Ca2+, and Mg2+ to the carboxylate group, cation-acetate binding affinities and binding modes remain actively debated. Here, we report the first use of Raman multivariate curve resolution (Raman-MCR) vibrational spectroscopy to obtain self-consistent free and bound metal acetate spectra and one-to-one binding constants, without the need to invoke any a priori assumptions regarding the shapes of the corresponding vibrational bands. The experimental results, combined with classical molecular dynamics simulations with a force field effectively accounting for electronic polarization via charge scaling and ab initio simulations, indicate that the measured binding constants pertain to direct (as opposed to water separated) ion pairing. The resulting binding constants do not scale with cation size, as the binding constant to Zn2+ is significantly larger than that to either Mg2+ or Ca2+, although Zn2+ and Mg2+ have similar radii that are about 25% smaller than Ca2+. Remaining uncertainties in the metal acetate binding free energies are linked to fundamental ambiguities associated with identifying the range of structures pertaining to non-covalently bound species.

Raman Optical Activity of Glucose and Sorbose in Extended Wavenumber Range.

Raman optical activity (ROA) is pursued as a promising method for structural analyses of sugars in aqueous solutions. In the present study, experimental Raman and ROA spectra of glucose and sorbose obtained in an extended range (50-4000 cm-1 ) are interpreted using molecular dynamics and density functional theory, with the emphasis on CH stretching modes. A reasonable theoretical basis for spectral interpretation was obtained already at the harmonic level. Anharmonic corrections led to minor shifts of band positions (up to 25 cm-1 ) below 2000 cm-1 , while the CH stretching bands shifted more, by ∼180 cm-1 , and better reproduced the experiment. However, the anharmonicities could be included on a relatively low approximation level only, and they did not always improve the harmonic band shapes. The dependence on the structure and conformation shows that the CH stretching ROA spectral pattern is a sensitive marker useful in saccharide structure studies.