Solid-state NMR Observables Research Articles

Comparative Study of REDOR and CPPI Derived Order Parameters by 1H-Detected MAS NMR and MD Simulations.

The measurement of dipolar couplings among directly bonded nuclei yields direct information on the amplitude of dynamic processes in the solid-state. For a reliable motional analysis using, e.g., the model-free approach, a correct quantification of the absolute values of these order parameters is absolutely essential. In the absence of a reference value for the rigid limit, too low dipolar coupling values might be misinterpreted as motion. Therefore, a detailed understanding of the effects that influence the quantification of the experimental order parameters is necessary. We compare here REDOR and CPPI derived order parameters assessed in 1H-detected experiments, and discuss the influence of remote protons and rf inhomogeneity on the extracted dipolar coupling constant for MAS rotation frequencies in the range 20-100 kHz. Experimental results are furthermore compared with the order parameter obtained from a molecular dynamics simulation. We find that fast magic-angle spinning up to 100 kHz can yield artifact-free REDOR based 1H,15N order parameters for perdeuterated and 100% amide back-exchanged proteins, and potentially even in uniformly protonated samples. We believe that awareness of systematic errors introduced by the measurement and in the analysis of order parameters will yield a better understanding of the dynamic properties of a protein derived from solid-state NMR observables.

Solid-State NMR-Restrained Ensemble Dynamics of a Membrane Protein in Explicit Membranes

Solid-state NMR has been used to determine the structures of membrane proteins in native-like lipid bilayer environments. Most structure calculations based on solid-state NMR observables are performed using simulated annealing with restrained molecular dynamics and an energy function, where all nonbonded interactions are represented by a single, purely repulsive term with no contributions from van der Waals attractive, electrostatic, or solvation energy. To our knowledge, this is the first application of an ensemble dynamics technique performed in explicit membranes that uses experimental solid-state NMR observables to obtain the refined structure of a membrane protein together with information about its dynamics and its interactions with lipids. Using the membrane-bound form of the fd coat protein as a model membrane protein and its experimental solid-state NMR data, we performed restrained ensemble dynamics simulations with different ensemble sizes in explicit membranes. For comparison, a molecular dynamics simulation of fd coat protein was also performed without any restraints. The average orientation of each protein helix is similar to a structure determined by traditional single-conformer approaches. However, their variations are limited in the resulting ensemble of structures with one or two replicas, as they are under the strong influence of solid-state NMR restraints. Although highly consistent with all solid-state NMR observables, the ensembles of more than two replicas show larger orientational variations similar to those observed in the molecular dynamics simulation without restraints. In particular, in these explicit membrane simulations, Lys40, residing at the C-terminal side of the transmembrane helix, is observed to cause local membrane curvature. Therefore, compared to traditional single-conformer approaches in implicit environments, solid-state NMR restrained ensemble simulations in explicit membranes readily characterize not only protein dynamics but also protein-lipid interactions in detail.

Influence of a Potentially Destabilizing Central Tryptophan on Transmembrane Helix Domains

Synthetic model peptides, such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide), provide a favorable “host” framework for investigations of the influence of chosen “guest” amino acids. For example, it is of interest to know the consequences of having a third, centrally located, tryptophan (Trp) within the hydrophobic core of a well characterized, anchored, transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). A Trp residue was introduced in the 12th or 13th position of GWALP23, and specific deuterated alanine labels (2H-Ala) were included as probes within the core helical sequence. The 2H quadrupolar splittings from solid-state NMR spectra of GWALP23-W12 and GWALP23-W13 show that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation (similar to GWALP23) in lipid bilayer membranes of DOPC, DLPC, and DMPC. Modified Gaussian and semi-static treatments of the dynamics yield similar conclusions. While a central Trp at position 12 or 13 does not alter the characteristics of bilayer-spanning GWALP23, incorporation of the peptide helix into the bilayer membrane becomes more difficult. The properties of W4, 5 GWALP23 are also being investigated, for comparison with the highly dynamic Y4, 5 and the less dynamic F4, 5 peptides. Deuterium labels at Ala3 and Ala21 will allow assessment of possible fraying of the ends of selected helices in differing lipid membrane environments.

Calcium-43 chemical shift and electric field gradient tensor interplay: a sensitive probe of structure, polymorphism, and hydration.

Calcium is the 5th most abundant element on earth, and is found in numerous biological tissues, proteins, materials, and increasingly in catalysts. However, due to a number of unfavourable nuclear properties, such as a low magnetogyric ratio, very low natural abundance, and its nuclear electric quadrupole moment, development of solid-state (43)Ca NMR has been constrained relative to similar nuclides. In this study, 12 commonly-available calcium compounds are analyzed via(43)Ca solid-state NMR and the information which may be obtained by the measurement of both the (43)Ca electric field gradient (EFG) and chemical shift tensors (the latter of which are extremely rare with only a handful of literature examples) is discussed. Combined with density functional theory (DFT) computations, this 'tensor interplay' is, for the first time for (43)Ca, illustrated to be diagnostic in distinguishing polymorphs (e.g., calcium formate), and the degree of hydration (e.g., CaCl2·2H2O and calcium tartrate tetrahydrate). For Ca(OH)2, we outline the first example of (1)H to (43)Ca cross-polarization on a sample at natural abundance in (43)Ca. Using prior knowledge of the relationship between the isotropic calcium chemical shift and the calcium quadrupolar coupling constant (CQ) with coordination number, we postulate the coordination number in a sample of calcium levulinate dihydrate, which does not have a known crystal structure. Natural samples of CaCO3 (aragonite polymorph) are used to show that the synthetic structure is present in nature. Gauge-including projector augmented-wave (GIPAW) DFT computations using accepted crystal structures for many of these systems generally result in calculated NMR tensor parameters which are in very good agreement with the experimental observations. This combination of (43)Ca NMR measurements with GIPAW DFT ultimately allows us to establish clear correlations between various solid-state (43)Ca NMR observables and selected structural parameters, such as unit cell dimensions and average Ca-O bond distances.

Influence of pH and Histidine Residues on Membrane-Spanning Helical Peptides

Synthetic model peptides such as GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide)provide a useful host framework for investigations of the influence of polar amino acids, for example histidine residues, within the hydrophobic core of a transmembrane helix. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-H12 reveal a marked difference in peptide behavior between acidic and neutral pH conditions. At neutral pH, GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes. To prevent the acid catalyzed degradation of lipids, we employed ether-linked DOPC bilayers to observe the effect of low pH on the L12H mutant. Under acidic conditions GWALP23-H12 is highly dynamic and exhibits multiple states. Indeed, the multi-state behavior of GWALP23-H12, when His is charged between pH 1.5 and pH 3, resembles closely that of GWALP23-R12 at neutral pH (J. Am. Chem. Soc. 132, 5803). The dramatic change in the behavior of GWALP23-H12 indicates a pKa value less than 3 for His near the center of a lipid bilayer. Investigations are in progress to chemically exchange the C2 imidazole hydrogen of His for deuterium in the peptide, toward a goal of enabling direct observation of the His ring by solid-state 2H NMR over a range of pH and buffer conditions.

Influence of a Central Tryptophan and of Cholesterol on the Properties of Defined Transmembrane Helical Peptides

GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) provides a favorable host framework for investigations of the influence of guest amino acids, for example a third, centrally located, Trp residue, within the hydrophobic core of a well characterized transmembrane helix. It is crucial to note that the orientation and rotation of GWALP23 are sensitive to single-residue replacements, in part because the membrane-spanning helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We introduced a Trp residue into position 12 or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra of GWALP23-W12 reveal that the peptide remains helical and retains a dominant preferred tilted transmembrane orientation with only a low extent of dynamic averaging, comparable to GWALP23 itself. Indeed, the tilt and dynamics of GWALP23-W12 are quite similar to the values observed for GWALP23 in DMPC bilayer membranes. We have analyzed the dynamics of the peptide helices using a modified Gaussian treatment as well as a semi-static treatment. The results indicate that a central Trp residue at position 12 does not appreciably perturb the properties of bilayer-spanning GWALP23. (By contrast, Arg-12 or Lys-12, when charged, induces multi-state behavior for GWALP23 in bilayer membranes [PNAS 110, 1692].) Additionally, we are investigating the influence of cholesterol upon the properties of membrane-spanning GWALP23, GWALP23-W12 and GWALP23-K12.

Single Tryptophan and Tyrosine Comparisons in the N-Terminal and C-Terminal Interface Regions of Transmembrane GWALP Peptides

Hydrophobic membrane-spanning helices often are flanked by interfacial aromatic or charged residues. In this paper, we compare the consequences of single Trp → Tyr substitutions at each interface for the properties of a defined transmembrane helix in the absence of charged residues. The choice of molecular framework is critical for these single-residue experiments because the presence of "too many" aromatic residues (more than one at either membrane-water interface) introduces excess dynamic averaging of solid state NMR observables. To this end, we compare the outcomes when changing W(5) or W(19), or both of them, to tyrosine in the well-characterized transmembrane peptide acetyl-GGALW(5)(LA)6LW(19)LAGA-amide ("GWALP23"). By means of solid-state (2)H and (15)N NMR experiments, we find that Y(19)GW(5)ALP23 displays similar magnitudes of peptide helix tilt as Y(5)GW(19)ALP23 and responds similarly to changes in bilayer thickness, from DLPC to DMPC to DOPC. The presence of Y(19) changes the azimuthal rotation angle ρ (about the helix axis) to a similar extent as Y(5), but in the opposite direction. When tyrosines are substituted for both tryptophans to yield GY(5,19)ALP23, the helix tilt angle is again of comparable magnitude, and furthermore, the preferred azimuthal rotation angle ρ is relatively unchanged from that of GW(5,19)ALP23. The extent of dynamic averaging increases marginally when Tyr replaces Trp. Yet, importantly, all members of the peptide family having single Tyr or Trp residues near each interface exhibit only moderate and not highly extensive dynamic averaging. The results provide important benchmarks for evaluating conformational and dynamic control of membrane protein function.

Influence of Histidine Residues on Transmembrane Helix Alignment

To investigate His residues in lipid bilayer membranes, we have employed GWALP23 (acetyl-GGALW5LALALALALALALW19LAGA-amide) as a favorable host peptide. Importantly, membrane-spanning GWALP23 is quite sensitive to single-residue replacements, in part because the transmembrane helix exhibits only limited dynamic averaging of solid-state NMR observables such as the 2H quadrupolar splitting (Biophys. J. 101, 2939). We inserted His residues into position 12 and/or 13 of GWALP23 (replacing either L12 or A13) and incorporated specific 2H-Ala labels within the helical core sequence. Solid-state 2H NMR spectra reveal a marked difference between the L12H mutant and the L12H-A13H double mutant. GWALP23-H12 exhibits a well-defined tilted transmembrane orientation in both DOPC and DLPC bilayer membranes, suggesting that the bilayer-incorporated histidine side chain is neutral (deprotonated) at an experimental pH of 5-6. By contrast, GWALP23-H12,13 is highly dynamic and exhibits multiple states which are in slow exchange on the NMR timescale. Indeed, the multi-state behavior of GWALP23-H12,13 between pH 4 and pH 9 resembles closely that of GWALP23-R12 (J. Am. Chem. Soc. 132, 5803). Further aspects of the pH dependence of transmembrane helices having one or two histidines are under investigation.

On the Combined Analysis of 2H and 15N/1H Solid-State NMR Data for Determination of Transmembrane Peptide Orientation and Dynamics

The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2H-alanine quadrupolar splittings together with 15N/1H dipolar couplings and 15N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA)6LWLAGA-amide). The results indicate that derivatives of GWALP23 incorporating diverse guest residues adopt a range of apparent tilt angles that span 5°–35° in lipid bilayer membranes. By comparing individual and combined analyses of specifically 2H- or 15N-labeled peptides incorporated in magnetically or mechanically aligned lipid bilayers, we examine the influence of data-set size/identity, and of explicitly modeled dynamics, on the deduced average orientations of the peptides. We conclude that peptides with small apparent tilt values (<∼10°) can be fitted by extensive families of solutions, which can be narrowed by incorporating additional 15N as well as 2H restraints. Conversely, peptides exhibiting larger tilt angles display more narrow distributions of tilt and rotation that can be fitted using smaller sets of experimental constraints or even with 2H or 15N data alone. Importantly, for peptides that tilt significantly more than 10° from the bilayer-normal, the contribution from rigid body dynamics can be approximated by a principal order parameter.

An ensemble dynamics approach to decipher solid-state NMR observables of membrane proteins

Solid-state NMR (SSNMR) is an invaluable tool for determining orientations of membrane proteins and peptides in lipid bilayers. Such orientational descriptions provide essential information about membrane protein functions. However, when a semi-static single conformer model is used to interpret various SSNMR observables, important dynamics information can be missing, and, sometimes, even orientational information can be misinterpreted. In addition, over the last decade, molecular dynamics (MD) simulation and semi-static SSNMR interpretation have shown certain levels of discrepancies in terms of transmembrane helix orientation and dynamics. Dynamic fitting models have recently been proposed to resolve these discrepancies by taking into account transmembrane helix whole body motions using additional parameters. As an alternative approach, we have developed SSNMR ensemble dynamics (SSNMR-ED) using multiple conformer models, which generates an ensemble of structures that satisfies the experimental observables without any fitting parameters. In this review, various computational methods for determining transmembrane helix orientations are discussed, and the distributions of VpuTM (from HIV-1) and WALP23 (a synthetic peptide) orientations from SSNMR-ED simulations are compared with those from MD simulations and semi-static/dynamic fitting models. Such comparisons illustrate that SSNMR-ED can be used as a general means to extract both membrane protein structure and dynamics from the SSNMR measurements. This article is part of a Special Issue entitled: Membrane protein structure and function.

Solid-State NMR Ensemble Dynamics as a Mediator between Experiment and Simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientations of membrane proteins and peptides in their native membrane bilayer environments. The deuterium ( 2H) quadrupolar splitting (DQS), one of the SSNMR observables, has been used to characterize the orientations of various single-pass transmembrane (TM) helices using a semistatic rigid-body model such as the geometric analysis of labeled alanine (GALA) method. However, dynamic information of these TM helices, which could be related to important biological function, can be missing or misinterpreted with the semistatic model. We have investigated the orientation of WALP23 in an implicit membrane of dimyristoylglycerophosphocholine by determining an ensemble of structures using multiple conformer models with a DQS restraint potential. When a single conformer is used, the resulting helix orientation (tilt angle ( τ) of 5.6 ± 3.2° and rotation angle ( ρ) of 141.8 ± 40.6°) is similar to that determined by the GALA method. However, as the number of conformers is increased, the tilt angles of WALP23 ensemble structures become larger (26.9 ± 6.7°), which agrees well with previous molecular dynamics simulation results. In addition, the ensemble structure distribution shows excellent agreement with the two-dimensional free energy surface as a function of WALP23's τ and ρ. These results demonstrate that SSNMR ensemble dynamics provides a means to extract orientational and dynamic information of TM helices from their SSNMR observables and to explain the discrepancy between molecular dynamics simulation and GALA-based interpretation of DQS data.

Transmembrane Helix Orientation and Dynamics: Insights from Ensemble Dynamics with Solid-State NMR Observables

As the major component of membrane proteins, transmembrane helices embedded in anisotropic bilayer environments adopt preferential orientations that are characteristic or related to their functional states. Recent developments in solid-state nuclear magnetic resonance (SSNMR) spectroscopy have made it possible to measure NMR observables that can be used to determine such orientations in a native bilayer environment. A quasistatic single conformer model is frequently used to interpret the SSNMR observables, but important motional information can be missing or misinterpreted in the model. In this work, we have investigated the orientation of the single-pass transmembrane domain of viral protein ”u“ (VpuTM) from HIV-1 by determining an ensemble of structures using multiple conformer models based on the SSNMR ensemble dynamics technique. The resulting structure ensemble shows significantly larger orientational fluctuations while the ensemble-averaged orientation is compatible with the orientation based on the quasistatic model. This observation is further corroborated by comparison with the VpuTM orientation from comparative molecular dynamics simulations in explicit bilayer membranes. SSNMR ensemble dynamics not only reveals the importance of transmembrane helix dynamics in interpretation of SSNMR observables, but also provides a means to simultaneously extract both transmembrane helix orientation and dynamics information from the SSNMR measurements.

Solid-State NMR Ensemble Dynamics as a Mediator between Experiment and Simulation

Solid-state NMR (SSNMR) is a powerful technique to describe the orientation of membrane proteins and peptides in their native membrane bilayer environments. Various SSNMR observables such as 2H NMR quadrupolar splitting (DQS), 15N chemical shift (CS), and 1H-15N dipolar coupling (DC) have been used to characterize the orientation of transmembrane (TM) helices with a static rigid-body model. In particular, TM helix orientational information is often related to membrane protein function and has been used to investigate the role of hydrophobic mismatch between TM helix and lipid bilayer. However, dynamic information of these TM helices can be missing or misinterpreted when a static model is used. We have investigated the orientation and dynamics of WALP23 in DMPC and GWALP23 in DLPC by determining ensemble of structures using multiple conformer models with various SSNMR restraint (DQS, CS, and DC) potentials. The resulting ensemble structures that satisfy the SSNMR observables are compared with those from MD simulations and free energy calculations in both explicit and implicit membrane. The good agreement illustrates that SSNMR-ensemble dynamics provides a means to extract the dynamic information from the SSNMR measurements and realistic explanation of the discrepancy between MD simulation and experimental interpretation based on a static model.

On the Treatment of Dynamics During Combined 2H GALA and 15N/ 1H PISEMA Analysis of Transmembrane Peptide Tilt using Solid-State NMR Data

Knowledge of transmembrane alpha-helix dynamics is important for interpretation of solid-state NMR observables. While precession of tilted peptides about the bilayer normal is commonly observed, additional dynamic features, such as anisotropic contributions from distributions of helix tilt or helix rotation, have the potential to influence the analysis. Previously, we compared independent analysis of 2H-alanine (“GALA”) and 15N/1H-backbone data sets (“PISEMA”) as constraints for determining helix tilt. Here we report combined analysis of 2H quadrupolar splittings together with 15N/1H dipolar couplings, using two methods to treat the dynamics, for the systematic evaluation of several membrane-spanning peptides based on the GWALP sequence (acetyl-GGALW(LA)6LWLAGA-amide), which tilt by 2°-30° in lipid bilayer membranes.By comparing individual and combined analyses of specifically 2H or 15N labeled peptides incorporated in mechanically or magnetically aligned lipid bilayers of differing thickness, we investigated the influence of data set size/identity, and of explicitly modeled dynamics, on the average apparent orientations of the peptides. We conclude that the peptides with small (less than ∼10°) apparent tilt values can be fitted by extensive collections of solutions, which can be narrowed by incorporating additional 15N as well as 2H restraints. Conversely, peptides that exhibit larger tilt angles have a narrower range of distributions of tilt and rotation that are consistent with the experimental data. The resulting smaller range can then be fitted using smaller sets of experimental constraints, or even with 2H or 15N data alone. Importantly, for the peptides that tilt significantly more than 10° from the bilayer normal, the contribution from rigid body dynamics can be approximated by a simple scaling factor (principal order parameter), and the concomitant apparent peptide orientation remains reasonably accurate.

Transmembrane Helix Orientation and Dynamics Examined by Ensemble Dynamics with Solid-State NMR Observables and Potential of Mean Force Calculations

Solid-state NMR (SSNMR) experiment is a very powerful technique to describe the orientation of membrane proteins and peptides in their native membrane bilayer environments. Such orientational description is invaluable since the function of membrane proteins and peptides involves a conformational change that often associates with their orientational changes. However, the present models used to interpret the SSNMR observables are rather static, and, in consequence, important motional information can be missing. In this work, we have investigated the orientation of single-pass transmembrane domain of virus protein “u” (Vpu) from HIV-1 by determining ensemble of structures using multiple conformer models. The resulting ensemble of structures showed significantly larger fluctuations in their orientation while averaged orientation maintained the compatible value with the static model. Further, we have compared the SSNMR ensemble dynamics results with the Vpu orientation from the molecular dynamics simulations and helix-tilt free energy calculations in explicit membranes.

Application of solid-state NMR restraint potentials in membrane protein modeling

We have developed a set of orientational restraint potentials for solid-state NMR observables including 15N chemical shift and 15N– 1H dipolar coupling. Torsion angle molecular dynamics simulations with available experimental 15N chemical shift and 15N– 1H dipolar coupling as target values have been performed to determine orientational information of four membrane proteins and to model the structures of some of these systems in oligomer states. The results suggest that incorporation of the orientational restraint potentials into molecular dynamics provides an efficient means to the determination of structures that optimally satisfy the experimental observables without an extensive geometrical search.