Methyl Group Dynamics Research Articles

Parsing Dynamics of Protein Backbone NH and Side-Chain Methyl Groups using Molecular Dynamics Simulations.

Experimental NMR spectroscopy and theoretical molecular dynamics (MD) simulations provide complementary insights into protein conformational dynamics and hence into biological function. The present work describes an extensive set of backbone NH and side-chain methyl group generalized order parameters for the Escherichia coli ribonuclease HI (RNH) enzyme derived from 2-μs microsecond MD simulations using the OPLS4 and AMBER-FF19SB force fields. The simulated generalized order parameters are compared with values derived from NMR 15N and 13CH2D spin relaxation measurements. The squares of the generalized order parameters, S2 for the N-H bond vector and Saxis2 for the methyl group symmetry axis, characterize the equilibrium distribution of vector orientations in a molecular frame of reference. Optimal agreement between simulated and experimental results was obtained by averaging S2 or Saxis2 calculated by dividing the simulated trajectories into 50 ns blocks (∼five times the rotational diffusion correlation time for RNH). With this procedure, the median absolute deviations (MAD) between experimental and simulated values of S2 and Saxis2 are 0.030 (NH) and 0.061 (CH3) for OPLS4 and 0.041 (NH) and 0.078 (CH3) for AMBER-FF19SB. The MAD between OPLS4 and AMBER-FF19SB are 0.021 (NH) and 0.072 (CH3). The generalized order parameters for the methyl group symmetry axis can be decomposed into contributions from backbone fluctuations, between-rotamer dihedral angle transitions, and within-rotamer dihedral angle fluctuations. Analysis of the simulation trajectories shows that (i) backbone and side chain conformational fluctuations exhibit little correlation and that (ii) fluctuations within rotamers are limited and highly uniform with values that depend on the number of dihedral angles considered. Low values of Saxis2, indicative of enhanced side-chain flexibility, result from between-rotamer transitions that can be enhanced by increased local backbone flexibility.

Coherences of Photoinduced Electron Spin Qubit Pair States in Photosystem I.

This publication presents the first comprehensive experimental study of electron spin coherences in photosynthetic reaction center proteins, specifically focusing on photosystem I (PSI). The ultrafast electron transfer in PSI generates spin-correlated radical pairs (SCRPs), which are entangled spin pairs formed in well-defined spin states (Bell states). Since their discovery in our group in the 1980s, SCRPs have been extensively used to enhance our understanding of structure-function relationships in photosynthetic proteins. More recently, SCRPs have been utilized as tools for quantum sensing. Electron spin decoherence poses a significant challenge in realizing practical applications of electron spin qubits, particularly the creation of quantum entanglement between multiple electron spins. This work is focused on the systematic characterization of decoherence in SCRPs of PSI. These decoherence times were measured as electron spin echo decay times, termed phase memory times (TM), at various temperatures. Decoherence was recorded on both transient SCRP states P700+A1- and thermalized states. Our study reveals that TM exhibits minimal dependence on the biological species, biochemical treatment, and paramagnetic species. The analysis indicates that nuclear spin diffusion and instantaneous diffusion mechanisms alone cannot explain the observed decoherence. As a plausible explanation we discuss the assumption that the low-temperature dynamics of methyl groups in the protein surrounding the unpaired electron spin centers is the main factor governing the loss of the spin coherence in PSI.

Probing Methyl Group Dynamics in Proteins by NMR Cross-Correlated Dipolar Relaxation and Molecular Dynamics Simulations.

Nuclear magnetic resonance (NMR) spin relaxation is the most informative approach to experimentally probe the internal dynamics of proteins on the picosecond to nanosecond time scale. At the same time, molecular dynamics (MD) simulations of biological macromolecules are steadily improving through better physical models, enhanced sampling methods, and increased computational power, and they provide exquisite information about flexibility and its role in protein stability and molecular interactions. Many examples have shown that MD is now adept in probing protein backbone motion, but improvements are still required toward a quantitative description of the dynamics of side chains, for example, probed by the dynamics of methyl groups. Thus far, the comparison of computation with experiment for side chain dynamics has primarily focused on the relaxation of 13C and 2H nuclei induced by autocorrelated variation of spin interactions. However, the cross-correlation of 13C-1H dipolar interactions in methyl groups offers an attractive alternative. Here, we establish a computational framework to extract cross-correlation relaxation parameters of methyl groups in proteins from all-atom MD simulations. To demonstrate the utility of the approach, cross-correlation relaxation rates of ubiquitin are computed from MD simulations performed with the AMBER99SB*-ILDN and CHARMM36 force fields. Simulation results were found to agree well with those obtained by experiment. Moreover, the data obtained with the two force fields are highly consistent.

The measurement of relaxation rates of degenerate 1H transitions in methyl groups of proteins using acute angle radiofrequency pulses

A simple NMR experiment for the measurement of transverse relaxation rates of degenerate 1H spins in 13CH3 methyl groups of deuterated proteins, is described. The experiment relies on the use of acute-angle 1H radio-frequency pulses, whereby a series of methyl 1H R2 decays is acquired with different values of the 1H pulse flip-angle, which modulates the relative contributions of the slow- and fast-relaxing components of 1H magnetization during the relaxation delay. These are subsequently analyzed simultaneously to extract the transverse relaxation rates of both components (RS and RF), along with the rate of cross-relaxation between these components, δ. The dipolar 1H–1H cross-correlated relaxation rate η = (RF – RS)/2 and the cross-relaxation rate δ, extracted from such acute-angle-pulse R2 decay series are compared with those derived from well-established methodology that uses relaxation-violated methyl 1H coherence transfer (so-called ‘forbidden’ experiments). Good agreement is achieved for the rates η derived from the two techniques, while slightly more accurate values of δ are obtained from analysis of acute-angle-pulse R2 series. Recording of acute-angle-pulse R2 series represents an attractive alternative to existing methodologies for quantifying methyl 1H relaxation and dynamics of the methyl three-fold symmetry axis in selectively [13CH3]-methyl-labeled, deuterated proteins – particularly so for very high-molecular-weight proteins, where the measurements of RF rates or the build-up of methyl 1H magnetization in relaxation-violated coherence transfer experiments, are problematic due to low sensitivity.

Low rotational barriers for the most dynamically active methyl groups in the proposed antiviral drugs for treatment of SARS-CoV-2, apilimod and tetrandrine

A recent screening study highlighted a molecular compound, apilimod, for its efficacy against the SARS-CoV-2 virus, while another compound, tetrandrine, demonstrated a remarkable synergy with the benchmark antiviral drug, remdesivir. Here, we find that because of significantly reduced potential energy barriers, which also give rise to pronounced quantum effects, the rotational dynamics of the most dynamically active methyl groups in apilimod and tetrandrine are much faster than those in remdesivir. Because dynamics of methyl groups are essential for biochemical activity, screening studies based on the computed potential energy profiles may help identify promising candidates within a given class of drugs.

Optimal design of adaptively sampled NMR experiments for measurement of methyl group dynamics with application to a ribosome-nascent chain complex

NMR measurements of cross-correlated nuclear spin relaxation provide powerful probes of polypeptide dynamics and rotational diffusion, free from contributions due to chemical exchange or interactions with external spins. Here, we report on the development of a sensitivity-optimized pulse sequence for the analysis of the differential relaxation of transitions within isolated 13CH3 spin systems, in order to characterise rotational diffusion and side chain order through the product S2τc. We describe the application of optimal design theory to implement a real-time ‘on-the-fly’ adaptive sampling scheme that maximizes the accuracy of the measured parameters. The increase in sensitivity obtained using this approach enables quantitative measurements of rotational diffusion within folded states of translationally-arrested ribosome–nascent chain complexes of the FLN5 filamin domain, and can be used to place strong limits on interactions between the domain and the ribosome surface.

Quantum Mechanical/Molecular Mechanical Simulations Model Retinal Dynamics during Membrane Activation of Rhodopsin

G-protein coupled receptors (GPCRs) - a superfamily of integral proteins that initiate signaling cascades upon ligand binding - are the most sought-after class of therapeutic targets. Solid-state 2H NMR spin relaxation times can be used to investigate the conformational dynamics in the membrane-bound state, but interpreting these data remains difficult. Here, in the case with the GPCR rhodopsin, we combine both quantum mechanics (QM) and molecular mechanics (MM) approaches [4] to study the rotational dynamics of the retinylidene methyl groups of the bound cofactor.

Hydration-Induced Disorder Lowers the Energy Barriers for Methyl Rotation in Drug Molecules.

The thermally activated dynamics of methyl groups are important for biochemical activity as they allow for a more efficient sampling of the energy landscape. Here, we compare methyl rotations in the dry and variously hydrated states of three primary drugs under consideration to treat the recent coronavirus disease (COVID-19), namely, hydroxychloroquine and its sulfate, dexamethasone and its sodium diphosphate, and remdesivir. We find that the main driving force behind the considerable reduction in the activation energy for methyl rotations in the hydrated state is the hydration-induced disorder in the methyl group local environments. Furthermore, the activation energy for methyl rotations in the hydration-induced disordered state is much lower than that in an isolated drug molecule, indicating that neither isolated molecules nor periodic crystalline structures can be used to analyze the potential landscape governing the side group dynamics in drug molecules. Instead, only the explicitly considered disordered structures can provide insight.

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

AbstractTo achieve efficient proton pumping in the light‐driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark‐adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side‐chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground‐state bR.

Dynamics of Bacteriorhodopsin in the Dark‐Adapted State from Solution Nuclear Magnetic Resonance Spectroscopy

To achieve efficient proton pumping in the light-driven proton pump bacteriorhodopsin (bR), the protein must be tightly coupled to the retinal to rapidly convert retinal isomerization into protein structural rearrangements. Methyl group dynamics of bR embedded in lipid nanodiscs were determined in the dark-adapted state, and were found to be mostly well ordered at the cytosolic side. Methyl groups in the M145A mutant of bR, which displays only 10 % residual proton pumping activity, are less well ordered, suggesting a link between side-chain dynamics on the cytosolic side of the bR cavity and proton pumping activity. In addition, slow conformational exchange, attributed to low frequency motions of aromatic rings, was indirectly observed for residues on the extracellular side of the bR cavity. This may be related to reorganization of the water network. These observations provide a detailed picture of previously undescribed equilibrium dynamics on different time scales for ground-state bR.

Site-specific dynamics of methyl groups probed by temporal evolution of heteronuclear 13C{1H} Overhauser polarisation encoded in 13C – 13C solid-state NMR correlation experiments

There is great interest in studying the dynamics of methyl groups which are sensitive probes of local packing and molecular motions in biosolids and proteins. In this work we show that the magnetisation created by transient heteronuclear 13C{1H} Overhauser effect can be exploited in two-dimensional 13C – 13C solid-state NMR correlation experiments for straightforward access to site-specific dynamics of methyl groups in fully protonated 13C labelled biomolecules. The methodology developed for this purpose is demonstrated on polycrystalline L-isoleucine and applied to probe residue-specific dynamics of methyl groups in the coat protein of Pf1 bacteriophage filamentous virus.

The influence of structure on the methyl group dynamics of polymorphic complexes: 6,6′-dimethyl-2,2′-dipyridyl with halo derivatives of benzoquinone acids

The structural analysis, neutron scattering, 1H NMR and computational methods combined to investigate new molecular complexes.

Opportunities and Challenges of Backbone, Sidechain, and RDC Experiments to Study Membrane Protein Dynamics in a Detergent-Free Lipid Environment Using Solution State NMR.

Whereas solution state NMR provided a wealth of information on the dynamics landscape of soluble proteins, only few studies have investigated membrane protein dynamics in a detergent-free lipid environment. Recent developments of smaller nanodiscs and other lipid-scaffolding polymers, such as styrene maleic acid (SMA), however, open new and promising avenues to explore the function-dynamics relationship of membrane proteins as well as between membrane proteins and their surrounding lipid environment. Favorably sized lipid-bilayer nanodiscs, established membrane protein reconstitution protocols and sophisticated solution NMR relaxation methods probing dynamics over a wide range of timescales will eventually reveal unprecedented lipid-membrane protein interdependencies that allow us to explain things we have not been able to explain so far. In particular, methyl group dynamics resulting from CEST, CPMG, ZZ exchange, and RDC experiments are expected to provide new and surprising insights due to their proximity to lipids, their applicability in large 100+ kDa assemblies and their simple labeling due to the availability of commercial precursors. This review summarizes the recent developments of membrane protein dynamics with a special focus on membrane protein dynamics in lipid-bilayer nanodiscs. Opportunities and challenges of backbone, side chain and RDC dynamics applied to membrane proteins are discussed. Solution-state NMR and lipid nanodiscs bear great potential to change our molecular understanding of lipid-membrane protein interactions.

Concomitant Polymorphism in an Organic Solid: Molecular and Crystal Structure and Intra- and Intermolecular Potential Contributions to tert-Butyl and Methyl Group Rotation.

We investigate the relationship between structure (crystal and molecular) and tert-butyl and methyl group dynamics in 2-(tert-butyl)-9-(4-(tert-butyl)phenyl)anthracene. Powder and single-crystal X-ray diffraction, taken together, show that different polycrystalline samples recrystallized from different solvents have different amounts of at least four polymorphs (crystallites having different crystal structures), of which we have identified three by single crystal X-ray diffraction. The molecules in the asymmetric units of the different crystal structures differ by the dihedral angle the tert-butylphenyl group makes with the anthracene moiety. Ab initio electronic structure calculations on the isolated molecule show that very little intramolecular energy is required to change this angle over a range of about 60° which is probably the origin of the concomitant polymorphism (crystals of more than one polymorph in a polycrystalline sample). Solid state 1 H nuclear magnetic resonance (NMR) spin-lattice relaxation experiments support the powder and single-crystal X-ray results and provide average NMR activation energies (closely related to rotational barriers) for the rotation of the tert-butyl groups and their constituent methyl groups. These barriers have both an intramolecular and an intermolecular component. The latter is sensitive to the crystal structure. The intramolecular components of the rotational barriers of the two tert-butyl groups in the isolated molecule are investigated with ab initio electronic structure calculations.

Complex Formation of the Tetracycline-Binding Aptamer Investigated by Specific Cross-Relaxation under DNP.

While dynamic nuclear polarization (DNP) under magic-angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid-state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) can be utilized to selectively obtain MAS-NMR spectra of an RNA aptamer in a tightly bound complex with a methyl-bearing ligand (tetracycline) due to the effective CH3 -reorientation at an optimized sample temperature of approximately 160 K. SCREAM-DNP can spectrally isolate the complex from non-bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM-DNP, between free tetracycline and RNA-bound tetracycline are discussed.

Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP

AbstractWhile dynamic nuclear polarization (DNP) under magic‐angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid‐state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross‐relaxation enhancement by active motions under DNP (SCREAM‐DNP) can be utilized to selectively obtain MAS‐NMR spectra of an RNA aptamer in a tightly bound complex with a methyl‐bearing ligand (tetracycline) due to the effective CH3‐reorientation at an optimized sample temperature of approximately 160 K. SCREAM‐DNP can spectrally isolate the complex from non‐bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM‐DNP, between free tetracycline and RNA‐bound tetracycline are discussed.

Methyl dynamics in amino acids modulate heteronuclear cross relaxation in the solid state under MAS DNP

Dynamic Nuclear Polarization (DNP) is a wide-spread technique for sensitivity enhancement of MAS NMR. During a typical MAS DNP experiment, several mechanisms resulting in polarization transfer may be active at the same time. One such mechanism which is most commonly active but up to now mostly disregarded is SCREAM-DNP (Specific Cross Relaxation Enhancement by Active Motions under DNP). This effect is generally observed in direct DNP experiments if molecular dynamics are supporting heteronuclear cross relaxation similar to the nuclear Overhauser effect. We investigate this effect for the CH3 groups of all methyl-bearing amino acids (i.e., alanine, valine, leucine, isoleucine, threonine, and methionine). At the typical DNP temperature of ∼110 K the three-fold reorientation dynamics are still active, and efficient SCREAM-DNP is observed. We discuss variations in enhancement factors obtained by this effect in context of sample temperature and sterical hindrance of the methyl group. Next to the direct transfer to the methyl carbon, we also find evidence for much weaker transfer from the methyl protons directly to other carbons in the amino acid molecule and succeed to correlate build-up dynamics with the CH dipole coupling which is modulated by the CH3 orientation. Besides methyl dynamics we also identify ring dynamics within proline as a source of SCREAM-DNP. Our results are the first step towards utilization of this effect as a specific probing techniqueusing methyl groups in protein systems.

Dynamics of Alanine Methyl Groups in Alanine Oligopeptides and Spider Dragline Silks with Different Packing Structures As Studied by 13C Solid-State NMR Relaxation

The dynamics of alanine methyl groups in alanine oligopeptides and polyalanine regions in Nephila clavata dragline and recombinant silks changed remarkably depending on the different packing arrangements. In particular, a notable difference in the 13C spin–lattice relaxation time T1 of Ala Cβ peaks was observed in the solid-state NMR between (Ala)6 and (Ala)7, which have rectangular and staggered packing arrangements, respectively. Moreover, the remarkably longer T1 value of the lowest field peak in Ala Cβ carbons clearly indicated the presence of staggered packing structure. Then, the relaxation behavior of polyalanine regions in spider dragline silks with mixed packing arrangements were clarified in both dry and hydrated states. The partial appearance of the staggered packing structure in (Ala)6 with rectangular packing structure was detected from the changes in line shapes of Ala 13Cβ peaks for the three kinds of 13C single-labeled (Ala)6 samples together with the change in their relaxation behavior.

Accurate Methyl Group Dynamics in Protein Simulations with AMBER Force Fields.

An approach is presented to directly simulate the dynamics of methyl groups in protein side-chains, as accessible via NMR spin relaxation measurements, by all-atom MD simulations. The method, which does not rely on NMR information or any system-specific adjustable parameters, is based on calculating the time-correlation functions (TCFs) of the C-H bonds in methyl groups and explicitly takes the truncation of the TCFs due to overall tumbling of the molecule into account. Using ubiquitin as a model protein, we show (i) that an accurate description of the methyl dynamics requires reparametrization of the potential energy barriers of methyl group rotation in the AMBER ff99SB*-ILDN force field (and related parameter sets), which was done with CCSD(T) coupled cluster calculations of isolated dipeptides as reference, and (ii) that the TIP4P/2005 solvation model yields overall tumbling correlation times that are in close agreement with experimental data. The methyl axis squared order parameters Saxis2 and associated correlation times τf, obtained within the Lipari-Szabo formalism, are in good agreement with the values derived from NMR deuterium relaxation experiments. Importantly, the relaxation rates and spectral densities derived from MD and NMR agree as well, enabling a direct comparison without assumptions inherent to simplified motional models.

NMR Relaxation and Molecular Dynamics Simulations of Side Chain Dynamics in Proteins

Molecular dynamics (MD) simulations and nuclear magnetic resonance (NMR) spin relaxation experiments have become increasingly powerful to study protein dynamics at atomic resolution due to steady improvements in physical models and computation power. Good agreement between generalized Lipari-Szabo (S2NH) order parameters derived from experiment and simulation is now observed for the backbone dynamics of a number of proteins. Unfortunately, the agreement for side chains, as e.g. probed by S2CH3 for methyl-containing side chains, is much poorer. In this work we discuss several issues with methyl side chains that need to be addressed to close the gap between NMR and MD. Accounting for protein tumbling is one very important factor to obtain a good agreement. In our hands, the application of improved water force fields with an appropriate way of including anisotropic overall protein tumbling improves the prediction of experimentally measured dynamic observables by MD simulations. We demonstrate these aspects for T4 lysozyme as a representative example. Our results guide the way for extracting from the NMR relaxation data the most accurate parameters that describe protein side chain dynamics and report on conformational entropy. Additionally, we reparametrized side chain dihedral angle energy barriers for methyl rotation in the Amber99SB∗-ILDN force field. We demonstrate that this leads to a much more realistic dynamics of methyl groups and is another important feature to close the gap between spectral densities from NMR relaxation and MD simulation.