Comparative analysis of the orientation of transmembrane peptides using solid-state 2H- and 15N-NMR: mobility matters (original) (raw)
Related papers
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.
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.
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.
Biophysical Journal, 2004
Solid-state NMR methods employing 2 H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane a-helical peptide acetyl-GWW(LA) 8 LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane a-helical conformation. A small tilt angle of 4.5°was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2°in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA) 6 LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.
Orientations of helical peptides in membrane bilayers by solid state NMR spectroscopy
Solid State Nuclear Magnetic Resonance, 1996
The orientations of helical peptides in membrane bilayers provide important structural information that is directly relevant to their functional roles, both alone and within the context of larger membrane proteins. The orientations can be readily determined with solid state NMR experiments on samples of "N-labeled peptides in lipid bilayers aligned between glass plates. The observed "N chemical shift frequencies can be directly interpreted to indicate whether the peptide's helix axis has a trans-membrane or an in-plane orientation. In order to distinguish between these possibilities on the basis of a single spectral parameter, e.g. the easily measured 15N chemical shift frequency, it is necessary to demonstrate that the secondary structure of the peptide is helical, generally by solution NMR spectroscopy of the same peptide in micelle samples, and that it is immobile in bilayers, generally from solid state NMR spectra of unoriented samples, Six different 20-30 residue peptides are shown to have orientations that fall into the categories of trans-membrane or in-plane helices. A model hydrophobic peptide was found to be trans-membrane, several different amphipathic helical peptides were found to have either trans-membrane or in-plane orientations, and a leader or signal peptide, generally regarded as hydrophobic, was found to have a significant population with an in-plane orientation.
Biophysical Journal, 2009
We report molecular dynamics simulations in the explicit membrane environment of a small membrane-embedded protein, sarcolipin, which regulates the sarcoplasmic reticulum Ca-ATPase activity in both cardiac and skeletal muscle. In its monomeric form, we found that sarcolipin adopts a helical conformation, with a computed average tilt angle of 28 5 6 and azymuthal angles of 66 5 22 , in reasonable accord with angles determined experimentally (23 5 2 and 50 5 4 , respectively) using solid-state NMR with separated-local-field experiments. The effects of time and spatial averaging on both 15 N chemical shift anisotropy and 1 H/ 15 N dipolar couplings have been analyzed using short-time averages of fast amide out-of-plane motions and following principal component dynamic trajectories. We found that it is possible to reproduce the regular oscillatory patterns observed for the anisotropic NMR parameters (i.e., PISA wheels) employing average amide vectors. This work highlights the role of molecular dynamics simulations as a tool for the analysis and interpretation of solid-state NMR data.
Journal of the American Chemical Society, 2008
Transmembrane-anchored WALP peptides of sequence acetyl-GWW(LA) n LWWA- [ethanol]amide-and the corresponding KALP peptides having KK instead of WW anchors -have been used extensively to characterize fundamental aspects of lipid/protein interactions. 1-4 Of particular interest has been the intrinsic tilt of these single-span α-helical peptides in hydrated lipid bilayer membranes. Consistently nonzero tilt angles (with respect to the bilayer normal) have been observed using solid-state 2 H NMR and the geometric analysis of labeled alanines (GALA) method. 5-7 The GALA analyses of the 19-residue WALP19 (n = 6) as well as WALP23 (n = 8), and KALP23, have indicated quite small average tilt angles τ in the range of about 4°−12° in several lipids. These experimental results have differed from predictions of much larger average tilt angles based upon molecular dynamics simulations. 8-10 A possible resolution to the discrepancy has entailed suggestions of averaging effects due to large-scale rotational motions of the peptide helices. 11,12
Order Parameters of a Transmembrane Helix in a Fluid Bilayer: Case Study of a WALP Peptide
Biophysical Journal, 2010
A new solid-state NMR-based strategy is established for the precise and efficient analysis of orientation and dynamics of transmembrane peptides in fluid bilayers. For this purpose, several dynamically averaged anisotropic constraints, including 13 C and 15 N chemical shift anisotropies and 13 C-15 N dipolar couplings, were determined from two different tripleisotope-labeled WALP23 peptides ( 2 H, 13 C, and 15 N) and combined with previously published quadrupolar splittings of the same peptide. Chemical shift anisotropy tensor orientations were determined with quantum chemistry. The complete set of experimental constraints was analyzed using a generalized, four-parameter dynamic model of the peptide motion, including tilt and rotation angle and two associated order parameters. A tilt angle of 21 was determined for WALP23 in dimyristoylphosphatidylcholine, which is much larger than the tilt angle of 5.5 previously determined from 2 H NMR experiments. This approach provided a realistic value for the tilt angle of WALP23 peptide in the presence of hydrophobic mismatch, and can be applied to any transmembrane helical peptide. The influence of the experimental data set on the solution space is discussed, as are potential sources of error.