Tilt and Azimuthal Angles of a Transmembrane Peptide: A Comparison between Molecular Dynamics Calculations and Solid-State NMR Data of Sarcolipin in Lipid Membranes (original) (raw)
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Biophysical journal, 2014
Solid-state NMR spectroscopy is emerging as a powerful approach to determine structure, topology, and conformational dynamics of membrane proteins at the atomic level. Conformational dynamics are often inferred and quantified from the motional averaging of the NMR parameters. However, the nature of these motions is difficult to envision based only on spectroscopic data. Here, we utilized restrained molecular dynamics simulations to probe the structural dynamics, topology and conformational transitions of regulatory membrane proteins of the calcium ATPase SERCA, namely sarcolipin and phospholamban, in explicit lipid bilayers. Specifically, we employed oriented solid-state NMR data, such as dipolar couplings and chemical shift anisotropy measured in lipid bicelles, to refine the conformational ensemble of these proteins in lipid membranes. The samplings accurately reproduced the orientations of transmembrane helices and showed a significant degree of convergence with all of the NMR pa...
Journal of Molecular Biology, 2006
Sarcolipin (SLN) is an integral membrane protein that is expressed in both skeletal and cardiac muscle, where it inhibits SERCA (calcium ATPase) by lowering its apparent Ca 2C affinity in a manner similar to that of its homologue phospholamban (PLN). We use solution NMR to map the structural changes occurring within SLN upon interaction with the regulatory target, SERCA, co-reconstituting the two proteins in dodecylphosphocholine (DPC) detergent micelles, a system that preserves the native structure of SLN and the activity of SERCA, with the goal of comparing these interactions with those of the previously studied PLN-SERCA complex. Our analysis of the structural dynamics of SLN in DPC micelles shows this polypeptide to be partitioned into four subdomains: a short unstructured N terminus (residues 1-6), a short dynamic helix (residues 7-14), a more rigid helix (residues 15-26), and an unstructured C terminus (residues 27-31). Upon addition of SERCA, the different domains behave according to their dynamics, molding onto the surface of the enzyme. Remarkably, each domain of SLN behaves in a manner similar to that of the corresponding domains in PLN, supporting the hypothesis that both SLN and PLN bind SERCA in the same groove and with similar mechanisms.
The Journal of Physical Chemistry B, 2011
We report an approach to determining membrane peptides and membrane protein complex structures by magic-angle-spinning solid-state NMR and molecular dynamics simulation. First, an ensemble of low energy structures of mastoparan-X, a wasp venom peptide, in lipid bilayers was generated by replica exchange molecular dynamics (REMD) simulation with the implicit membrane/solvent model. Next, peptide structures compatible with experimental 13 C R , C β , and C 0 chemical shifts were selected from the ensemble. The 13 C R chemical shifts alone were sufficient for the selection with backbone rmsd's of ∼0.8 Å from the experimentally determined structure. The dipolar couplings between the peptide protons and lipid 2 H/ 31 P nuclei were obtained from the 13 C-observed 2 H/ 31 P-selective 1 H-demagnetization experiments for selecting the backbone and side chain structures relative to the membrane. The simulated structure agreed with the experimental one in the depth and orientation. The REMD simulation can be used for supplementing the limited structural constraints obtainable from the solid-state NMR spectra.
Orientation and Dynamics of Peptides in Membranes Calculated from 2H-NMR Data
Biophysical Journal, 2009
Solid-state 2 H-NMR is routinely used to determine the alignment of membrane-bound peptides. Here we demonstrate that it can also provide a quantitative measure of the fluctuations around the distinct molecular axes. Using several dynamic models with increasing complexity, we reanalyzed published 2 H-NMR data on two representative a-helical peptides: 1), the amphiphilic antimicrobial peptide PGLa, which permeabilizes membranes by going from a monomeric surface-bound to a dimeric tilted state and finally inserting as an oligomeric pore; and 2), the hydrophobic WALP23, which is a typical transmembrane segment, although previous analysis had yielded helix tilt angles much smaller than expected from hydrophobic mismatch and molecular dynamics simulations. Their 2 H-NMR data were deconvoluted in terms of the two main helix orientation angles (representing the time-averaged peptide tilt and azimuthal rotation), as well as the amplitudes of fluctuation about the corresponding molecular axes (providing the dynamic picture). The mobility of PGLa is found to be moderate and to correlate well with the respective oligomeric states. WALP23 fluctuates more vigorously, now in better agreement with the molecular dynamics simulations and mismatch predictions. The analysis demonstrates that when 2 H-NMR data are fitted to extract peptide orientation angles, an explicit representation of the peptide rigid-body angular fluctuations should be included.
Biophysical Journal, 2011
The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2 H-alanine quadrupolar splittings together with 15 N/ 1 H dipolar couplings and 15 N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA) 6 LWLAGA-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 2 H-or 15 N-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 15 N as well as 2 H 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 2 H or 15 N 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.
Solid state NMR analysis of peptides in membranes: Influence of dynamics and labeling scheme
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
The functional state of a membrane-active peptide is often defined by its conformation, molecular orientation, and its oligomeric state in the lipid bilayer. These "static" structural properties can be routinely studied by solid state NMR using isotope-labeled peptides. In the highly dynamic environment of a liquid crystalline biomembrane, however, the whole-body fluctuations of a peptide are also of paramount importance, although difficult to address and most often ignored. Yet it turns out that disregarding such motional averaging in calculating the molecular alignment from orientational NMR-constraints may give a misleading, if not false picture of the system. Here, we demonstrate that the reliability of a simplified static or an advanced dynamic data analysis depends critically on the choice of isotope labeling scheme used. Two distinctly different scenarios have to be considered. When the labels are placed on the side chains of a helical peptide (such as a CD 3-or CF 3-group attached to the C α \C β bond), their nuclear spin interaction tensors are very sensitive to motional averaging. If this effect is not properly accounted for, the helix tilt angle tends to be severely underestimated. At the same time, the analysis of labels in the side chains allows to extract valuable dynamical information about whole-body fluctuations of the peptide helix in the membrane. On the other hand, the alternative labeling scheme where 15 N-labels are accommodated within the peptide backbone, will yield nearly correct helix tilt angles, irrespective as to whether dynamics are taken into account or not.
Solid-state NMR approaches to measure topological equilibria and dynamics of membrane polypeptides
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2010
Biological membranes are characterized by a high degree of dynamics. In order to understand the function of membrane proteins and even more of membrane-associated peptides, these motional aspects have to be taken into consideration. Solid-state NMR spectroscopy is a method of choice when characterizing topological equilibria, molecular motions, lateral and rotational diffusion as well as dynamic oligomerization equilibria within fluid phase lipid bilayers. Here we show and review examples where the 15 N chemical shift anisotropy, dipolar interactions and the deuterium quadrupolar splittings have been used to analyze motions of peptides such as peptaibols, antimicrobial sequences, Vpu, phospholamban or other channel domains. In particular, simulations of 15 N and 2 H-solid-state NMR spectra are shown of helical domains in uniaxially oriented membranes when rotation around the membrane normal or the helix long axis occurs.
Biophysical Journal, 2009
Membrane proteins and peptides exhibit a preferred orientation in the lipid bilayer while fluctuating in an anisotropic manner. Both the orientation and the dynamics have direct functional implications, but motions are usually not accessible, and structural descriptions are generally static. Using simulated data, we analyze systematically the impact of whole-body motions on the peptide orientations calculated from two-dimensional polarization inversion spin exchange at the magic angle (PISEMA) NMR. Fluctuations are found to have a significant effect on the observed spectra. Nevertheless, wheel-like patterns are still preserved, and it is possible to determine the average peptide tilt and azimuthal rotation angles using simple static models for the spectral fitting. For helical peptides undergoing large-amplitude fluctuations, as in the case of transmembrane monomers, improved fits can be achieved using an explicit dynamics model that includes Gaussian distributions of the orientational parameters. This method allows extracting the amplitudes of fluctuations of the tilt and azimuthal rotation angles. The analysis is further demonstrated by generating first a virtual PISEMA spectrum from a molecular dynamics trajectory of the model peptide, WLP23, in a lipid membrane. That way, the dynamics of the system from which the input spectrum originates is completely known at atomic detail and can thus be directly compared with the dynamic output obtained from the fit. We find that fitting our dynamics model to the polar index slant angles wheel gives an accurate description of the amplitude of underlying motions, together with the average peptide orientation.
Journal of Chemical Theory and Computation, 2018
The membrane alignment of helical amphiphilic peptides in oriented phospholipid bilayers can be obtained as ensemble and time averages from solid state 2 H NMR by fitting the quadrupolar splittings to ideal α-helices. At the same time, molecular dynamics (MD) simulations can provide atomistic insight into peptide−membrane systems. Here, we evaluate the potential of MD simulations to complement the experimental NMR data that is available on three exemplary systems: the natural antimicrobial peptide PGLa and the two designer-made peptides MSI-103 and KIA14, whose sequences were derived from PGLa. Each peptide was simulated for 1 μs in a DMPC lipid bilayer. We calculated from the MD simulations the local angles which define the side chain geometry with respect to the peptide helix. The peptide orientation was then calculated (i) directly from the simulation, (ii) from back-calculated MD-derived NMR splittings, and (iii) from experimental 2 H NMR splittings. Our findings are that (1) the membrane orientation and secondary structure of the peptides found in the NMR analysis are generally well reproduced by the simulations; (2) the geometry of the side chains with respect to the helix backbone can deviate significantly from the ideal structure depending on the specific residue, but on average all side chains have the same orientation; and (3) for all of our peptides, the azimuthal rotation angle found from the MDderived splittings is about 15°smaller than the experimental value.
Biophysical Journal, 2008
Molecular dynamics simulations and 31 P-NMR spin-lattice (R 1 ) relaxation rates from 0.022 to 21.1 T of fluid phase dipalmitoylphosphatidylcholine bilayers are compared. Agreement between experiment and direct prediction from simulation indicates that the dominant slow relaxation (correlation) times of the dipolar and chemical shift anisotropy spin-lattice relaxation are ;10 ns and 3 ns, respectively. Overall reorientation of the lipid body, consisting of the phosphorus, glycerol, and acyl chains, is well described within a rigid-body model. Wobble, with D ? ¼ 1-2 3 10 8 s À1 , is the primary component of the 10 ns relaxation; this timescale is consistent with the tumbling of a lipid-sized cylinder in a medium with the viscosity of liquid hexadecane. The value for D k ; the diffusion constant for rotation about the long axis of the lipid body, is difficult to determine precisely because of averaging by fast motions and wobble; it is tentatively estimated to be 1 3 10 7 s À1 . The resulting D k /D ? % 0.1 implies that axial rotation is strongly modulated by interactions at the lipid/water interface. Rigid-body modeling and potential of mean force evaluations show that the choline group is relatively uncoupled from the rest of the lipid. This is consistent with the ratio of chemical shift anisotropy and dipolar correlation times reported here and the previous observations that 31 P-NMR lineshapes are axially symmetric even in the gel phase of dipalmitoylphosphatidylcholine.