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1.
Solid-state NMR and hydrogen-deuterium exchange in a bilayer-solubilized peptide: structural and mechanistic implications
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Hydrogen-deuterium exchange has been monitored by solid-state NMR to investigate the structure of gramicidin M in a lipid bilayer and to investigate the mechanisms for polypeptide insertion into a lipid bilayer. Through exchange it is possible to observe 15N-2H dipolar interactions in oriented samples that yield precise structural constraints. In separate experiments the pulse sequence SFAM was used to measure dipolar distances in this structure, showing that the dimer is antiparallel. The combined use of orientational and distance constraints is shown to be a powerful structural approach. By monitoring the hydrogen-deuterium exchange at different stages in the insertion of peptides into a bilayer environment it is shown that dimeric gramicidin is inserted into the bilayer intact, i.e., without separating into monomer units. The exchange mechanism is investigated for various sites and support for a relayed imidic acid mechanism is presented. Both acid and base catalyzed mechanisms may be operable. The nonexchangeable sites clearly define a central core to which water is inaccessible or hydroxide or hydronium ion is not even momentarily stable. This provides strong evidence that this is a nonconducting state. 相似文献
2.
Direct measurement of 1H-1H dipolar couplings in proteins: A complement to traditional NOE measurements 总被引:1,自引:0,他引:1
Tian F Fowler CA Zartler ER Jenney FA Adams MW Prestegard JH 《Journal of biomolecular NMR》2000,18(1):23-31
An intensity-based constant-time COSY (CT-COSY) method is described for measuring 1H-1H residual dipolar couplings of proteins in weakly aligned media. For small proteins, the overall sensitivity of this experiment is comparable to the NOESY experiment. In cases where the 1H-1H distances are defined by secondary structure, such as 1H-1HN and 1HN-1HN sequential distances in -helices and -sheets, these measurements provide useful orientational constraints for protein structure determination. This experiment can also be used to provide distance information similar to that obtained from NOE connectivities once the angular dependence is removed. Because the measurements are direct and non-coherent processes, such as spin diffusion, do not enter, the measurements can be more reliable. The 1/r
3 distance dependence of directly observed dipolar couplings, as compared with the 1/r
6 distance dependence of NOEs, also can provide longer range distance information at favorable angles. A simple 3D, 15N resolved version of the pulse sequence extends the method to provide the improved resolution required for application to larger biomolecules. 相似文献
3.
Jesús Salgado Stephan L. Grage Leslie H. Kondejewski Robert S. Hodges Ronald N. McElhaney Anne S. Ulrich 《Journal of biomolecular NMR》2001,21(3):191-208
The antimicrobial properties of the cyclic -sheet peptide gramicidin S are attributed to its destabilizing effect on lipid membranes. Here we present the membrane-bound structure and alignment of a derivative of this peptide, based on angular and distance constraints. Solid-state 19F-NMR was used to study a 19F-labelled gramicidin S analogue in dimyristoylphosphatidylcholine bilayers at a lipid:peptide ratio of 80:1 and above. Two equivalent leucine side chains were replaced by the non-natural amino acid 4F-phenylglycine, which serves as a highly sensitive reporter on the structure and dynamics of the peptide backbone. Using a modified CPMG multipulse sequence, the distance between the two 19F-labels was measured from their homonuclear dipolar coupling as 6 Å, in good agreement with the known backbone structure of natural gramicidin S in solution. By analyzing the anisotropic chemical shift of the 19F-labels in macroscopically oriented membrane samples, we determined the alignment of the peptide in the bilayer and described its temperature-dependent mobility. In the gel phase, the 19F-labelled gramicidin S is aligned symmetrically with respect to the membrane normal, i.e., with its cyclic -sheet backbone lying flat in the plane of the bilayer, which is fully consistent with its amphiphilic character. Upon raising the temperature to the liquid crystalline state, a considerable narrowing of the 19F-NMR chemical shift dispersion is observed, which is attributed the onset of global rotation of the peptide and further wobbling motions. This study demonstrates the potential of the 19F nucleus to describe suitably labelled polypeptides in membranes, requiring only little material and short NMR acquisition times. 相似文献
4.
Summary The complete structure determination of a polypeptide in a lipid bilayer environment is demonstrated built solely upon orientational constraints derived from solid-state NMR observations. Such constraints are obtained from isotopically labeled samples uniformly aligned with respect to the B0 field. Each observation constrains the molecular frame with respect to B0 and the bilayer normal, which are arranged to be parallel. These constraints are not only very precise (a few tenths of a degree), but also very accurate. This is clearly demonstrated as the back bone structure is assembled sequentially and the i to i+6 hydrogen bonds in this structure of the gramicidin channel are shown on average to be within 0.5 Å of ideal geometry. Similarly, the side chains are assembled independently and in a radial direction from the backbone. The lack of considerable atomic overlap between side chains also demonstrates the accuracy of the constraints. Through this complete structure, solid-state NMR is demonstrated as an approach for determining three-dimensional macromolecular structure. 相似文献
5.
Green sulfur photosynthetic bacteria optimize their antennas, chlorosomes, especially for harvesting weak light by organizing bacteriochlorophyll (BChl) assembly without any support of proteins. As it is difficult to crystallize the organelles, a high-resolution structure of the light-harvesting devices in the chlorosomes has not been clarified. We have determined the structure of BChl c assembly in the intact chlorosomes from Chlorobium limicola on the basis of 13C dipolar spin-diffusion solid-state NMR analysis of uniformly 13C-labeled chlorosomes. About 90 intermolecular C–C distances were obtained by the simultaneous assignment of distance correlations and the structure optimization preceded by the polarization-transfer matrix analysis. An atomic structure was obtained, using these distance constraints. The determined structure of the chlorosomal BChl c assembly is built with the parallel layers of piggyback-dimers. This supramolecular structure would provide insights into the mechanism of weak-light capturing. 相似文献
6.
P. Berthault N. Birlirakis G. Rubinstenn P. Sinaÿ H. Desvaux 《Journal of biomolecular NMR》1996,8(1):23-35
Summary A recent 1H NMR method has been applied to the determination of the solution structure and internal dynamics of a synthetic mixed C/O trisaccharide related to sialyl Lewisx. Varying the rf field offset in ROESY-type experiments enabled the measurement of longitudinal and transverse dipolar cross-relaxation rates with high accuracy. Assuming that for each proton pair the motion could be represented by a single exponential autocorrelation function, it was possible to derive geometrical parameters (r) and dynamic parameters cp. With this assumption, 224 cross-relaxation rates have been transformed into 30 interproton distance constraints and 30 dipolar correlation times. The distance constraints have been used in a simulated-annealing procedure. This trisaccharide exhibits a structure close to the O-glycosidic analogue, but its flexibility seems highly reduced. On the basis of the determined structure and dynamics, it is shown that no conformational exchange occurs, the molecule existing in the form of a unique family in aqueous solution. In order to assess the quality of the resulting structures and to validate this new experimental procedure of distance extraction, we finally compare these solution structures to the ones obtained using three different sets of distances deduced from three choices of internal reference. It appears that this procedure allows the determination of the most precise and accurate solution.Abbreviations COSY
correlation spectroscopy
- NOE
nuclear Overhauser enhancement
- NOESY
nuclear Overhauser enhancement spectroscopy; rmsd, root-mean-square deviation
- ROESY
rotating frame Overhauser enhancement spectroscopy
- SLex
sialyl Lewisx
- TOCSY
total correlation spectroscopy 相似文献
7.
Orientational constraints as three-dimensional structural constraints from chemical shift anisotropy: the polypeptide backbone of gramicidin A in a lipid bilayer. 总被引:1,自引:1,他引:1
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W. Mai W. Hu C. Wang T. A. Cross 《Protein science : a publication of the Protein Society》1993,2(4):532-542
Chemical shifts observed from samples that are uniformly aligned with respect to the magnetic field can be used as very high-resolution structural constraints. This constraint takes the form of an orientational constraint rather than the more familiar distance constraint. The accuracy of these constraints is dependent upon the quality of the tensor characterization. Both tensor element magnitudes and tensor orientations with respect to the molecular frame need to be considered. Here these constraints have been used to evaluate models for the channel conformation of gramicidin A. Of the three models used, the one experimentally derived model of gramicidin in sodium dodecyl sulfate micelles fits the data least well. 相似文献
8.
The ratio of the internucleotide dipolar coupling and the corresponding one-bond imino 15N-1H dipolar coupling provides a measure for the N···H/H-N distance ratio. Measurements were carried out for a dodecamer, d(CGCGAATTCGCG)2, in which a C-G and an A-T basepair were uniformly enriched in 15N. When assuming H-bonds to be perfectly linear, dipolar data indicate time-averaged hydrogen bond lengths of 1.80±0.03 Å for A-T and 1.86±0.02 Å for C-G. When using H-bond orientations from high resolution X-ray data, H-bond lengths are about 0.1 Å shorter. 相似文献
9.
Residual heteronuclear dipolar couplings obtained from partially oriented protein samples can provide unique NMR constraints for protein structure determination. However, partial orientation of protein samples also causes severe 1 H line broadening resulting from residual 1 H-1H dipolar couplings. In this communication we show that band-selective 1H homonuclear decoupling during data acquisition is an efficient way to suppress residual 1H-1H dipolar couplings, resulting in spectra that are still amenable to solution NMR analysis, even with high degrees of alignment. As an example, we present a novel experiment with improved sensitivity for the measurement of one-bond 1 HN-15N residual dipolar couplings in a protein sample dissolved in magnetically aligned liquid crystalline bicelles. 相似文献
10.
Rodolfo R. Biekofsky Frederick W. Muskett Jürgen M. Schmidt Stephen R. Martin J. Peter Browne Peter M. Bayley James Feeney 《FEBS letters》1999,460(3):4403
This work shows that the partial replacement of diamagnetic Ca2+ by paramagnetic Tb3+ in Ca2+/calmodulin systems in solution allows the measurement of interdomain NMR pseudocontact shifts and leads to magnetic alignment of the molecule such that significant residual dipolar couplings can be measured. Both these parameters can be used to provide structural information. Species in which Tb3+ ions are bound to only one domain of calmodulin (the N-domain) and Ca2+ ions to the other (the C-domain) provide convenient systems for measuring these parameters. The nuclei in the C-domain experience the local magnetic field induced by the paramagnetic Tb3+ ions bound to the other domain at distances of over 40 Å from the Tb3+ ion, shifting the resonances for these nuclei. In addition, the Tb3+ ions bound to the N-domain of calmodulin greatly enhance the magnetic susceptibility anisotropy of the molecule so that a certain degree of alignment is produced due to interaction with the external magnetic field. In this way, dipolar couplings between nuclear spins are not averaged to zero due to solution molecular tumbling and yield dipolar coupling contributions to, for example, the one-bond 15N-1H splittings of up to 17 Hz in magnitude. The degree of alignment of the C-domain will also depend on the degree of orientational freedom of this domain with respect to the N-domain containing the Tb3+ ions. Pseudocontact shifts for NH groups and 1H-15N residual dipolar couplings for the directly bonded atoms have been measured for calmodulin itself, where the domains have orientational freedom, and for the complex of calmodulin with a target peptide from skeletal muscle myosin light chain kinase, where the domains have fixed orientations with respect to each other. The simultaneous measurements of these parameters for systems with domains in fixed orientations show great potential for the determination of the relative orientation of the domains. 相似文献
11.
The assignment of the 1H and 15Nnuclear magnetic resonance spectra of the Src-homology region 3 domain ofchicken brain -spectrin has been obtained. A set of solutionstructures has been determined from distance and dihedral angle restraints,which provide a reasonable representation of the protein structure insolution, as evaluated by a principal component analysis of the globalpairwise root-mean-square deviation (rmsd) in a large set of structuresconsisting of the refined and unrefined solution structures and the crystalstructure. The solution structure is well defined, with a lower degree ofconvergence between the structures in the loop regions than in the secondarystructure elements. The average pairwise rmsd between the 15 refinedsolution structures is 0.71 ± 0.13 Å for the backbone atoms and1.43 ± 0.14 Å for all heavy atoms. The solution structure isbasically the same as the crystal structure. The average rmsd between the 15refined solution structures and the crystal structure is 0.76 Å forthe backbone atoms and 1.45 ± 0.09 Å for all heavy atoms. Thereare, however, small differences probably caused by intermolecular contactsin the crystal structure. 相似文献
12.
S.V. Sychev N.A. Nevskaya St. Jordanov E.N. Shepel A.I. Miroshnikov V.T. Ivanov 《Bioorganic chemistry》1980,9(1):121-151
The conformational states in dioxane and ethanol of gramicidin A and of analogs varying in chain length and amino acid sequence have been studied. Infrared, CD, and polarization of fluorescence spectra of the peptides were measured, from which dimerization constants were determined and spectral characteristics of the monomeric and dimeric states obtained. Resonance splitting of the amide I ir band has been calculated for all gramicidin A models proposed earlier. Detailed comparison of the experimental and computed spectra showed that the four dimeric gramicidin species present in solution are predominantly antiparallel double ?ππld helices in equilibrium with smaller amounts of head-to-head associated πLD helices. The gramicidin A monomer was found to be a πLD4.4 helix in dioxane. For each conformational form the number of residues per turn and the helical sense were determined. The relationship between the amino acid sequence and the structure and stability of the dimer in the series of gramicidin A and its analogs is discussed. The above findings are rationalized in terms of the membrane channel properties of gramicidin A, in particular the conformational rearrangements occurring during the passage of metal ions through the channel and also the differences in conformation of the antibiotic in nonpolar solutions and in the membrane. 相似文献
13.
Historically, the task of determining the structure of membrane proteins has been hindered by experimental difficulties associated with their lipid-embedded domains. Here, we provide an overview of recently developed experimental and predictive tools that are changing our view of this largely unexplored territory - the 'Wild West' of structural biology. Crystallography, single-particle methods and atomic force microscopy are being used to study huge membrane proteins with increasing detail. Solid-state nuclear magnetic resonance strategies provide orientational constraints for structure determination of transmembrane (TM) alpha-helices and accurate measurements of intramolecular distances, even in very complex systems. Longer distance constraints are determined by site-directed spin-labelling electron paramagnetic resonance, but current labelling strategies still constitute some limitation. Other methods, such as site-specific infrared dichroism, enable orientational analysis of TM alpha-helices in aligned bilayers and, combined with novel computational and predictive tools that use evolutionary conservation data, are being used to analyze TM alpha-helical bundles. 相似文献
14.
A model based on the solution of the electrostatic potential for a geometry of three dielectric regions associated with a gramicidin A channel (GA) is presented. The model includes a cylindrical dielectric layer to represent the peptide backbone and dipole rings to account for dipolar side chains. Image potential and dipolar contributions for different orientations and positions along the channel are analyzed. The conductance of GA and two analogues obtained by substituting the amino acid at position 1 are studied. The numerical simulation reproduces experimental results (Barrett et al. 1986, Biophys J 49, 673–686) and supports the idea that electrostatic dipole-ion interactions are of primary importance in gramicidin channel function.
Correspondence to: G. Martinez 相似文献
15.
Gramicidin cation channel: an experimental determination of the right-handed helix sense and verification of beta-type hydrogen bonding 总被引:16,自引:0,他引:16
Due to the difficulty of obtaining protein/lipid cocrystals for diffraction studies, structural research on intrinsic membrane proteins and polypeptides has been largely restricted to indirect experimental techniques. Hence, many fundamental questions associated with peptide/lipid systems remain unanswered. In particular, the handedness of the gramicidin A transmembrane ion channel incorporated into lipid bilayers has been an open question for nearly two decades. In this study, solid-state 15N NMR spectroscopy is employed to probe directly the secondary structure of the polypeptide backbone. Recent determinations of the 15N chemical shift anisotropy tensor with respect to the molecular frame enable the quantitative evaluation of the 15N chemical shift resonances obtained from oriented dimyristoylphosphatidylcholine (DMPC) bilayer samples containing specific site 15N labeled gramicidin. This direct structural approach verifies the beta-sheet hydrogen-bonding pattern proposed by Urry [Urry, D. W. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 672-676] and determines that in our DMPC bilayer preparations the gramicidin channel is right-handed. Additional structural information is provided by the 15N chemical shift data in the form of orientational constraints on the C alpha-C alpha axis orientation of individual peptides relative to the helix axis. The significance of these solid-state NMR results lies in the direct determination of the helix sense and the verification of the beta-type hydrogen bonding, in the development of the solid-state NMR methods for obtaining such information, and in emphasizing the importance of having direct structural data at atomic resolution. 相似文献
16.
《Journal of molecular biology》1996,264(4):757-769
The structure of a parallel left-handed double-helical form of gramicidin was detected by circular dichroism spectroscopy and determined using 500 and 600 MHz NMR in CaCl2/methanol solution. Measurements of TOCSY, DQF-COSY and NOESY spectra were converted into 604 distance and 48 torsional angle constraints for structure calculations. Stereospecific assignments and χ1angles were calculated using3Jαβ, dαβ(i,i), dNβ(i,i) and dNγ(i,i). χ2angles were determined using dαβ(i,i), dNβ(i,i), dβδ(i,i), dNγ(i,i) and dαγ(i,i). The calculations of initial structures were performed using the distance geometry/simulated annealing method in XPLOR. The initial structures were further refined and energy minimized using simulated annealing/molecular dynamics methods. Back-calculations for every generated structure were also performed to check their consistency with the experimental data.187 final structures with no violations above the threshold conditions (0.05 Å, 5°, 5°, 0.5 Å and 5° for bonds, angles, improper, NOE and cdihe, respectively) were produced from the 200 initial structures. Twenty structures with the lowest NOE energies were used for further analysis. The average r.m.s. deviations for the 20 structures are 0.64 Å for backbone and 1.1 Å for all non-hydrogen atoms.Gramicidin in this form, with approximately 5.7 residues per turn, is a parallel double helical dimer. The length along the helix axis is about 30 Å and the inner pore diameter varies from 1 to 2 Å. It is different from all other gramicidin structures determined to date. The presence of Ca2 +stabilises a conformation that prevents the binding of monovalent cations. It is likely that this structure is related to a non-channel, antibiotic role of gramicidin. 相似文献
17.
A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring 15N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral
overlap. Measurement of backbone 15N R
1, R
1ρ, 15N–{1H} NOE, and 15N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium
channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane
domain, KcsATM, exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, τc = 38 ± 2.5 ns, at 50 °C and a diffusion anisotropy, , corresponding to an aspect ratio a/b ≥ 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S
2 values in the 0.2–0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils.
Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this
amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence
of elevated backbone dynamics on the ps–ns time scale for the 5-residue selectivity filter, which selects K+ ions to enter the channel.
Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .
An erratum to this article can be found at 相似文献
18.
Summary
15N-C and15N-C J couplings were measured for the backbone of staphylococcal nuclease, uniformly enriched with15N and13C. It is found that theIJC'N coupling is similar for -sheet, J=14.8 ± 0.5 and for -helix, J = 14.8 ± 0.4 but tends to be larger for the unstructured N- and C-terminal ends of the protein (J=15.6 ± 0.5). On average,1JNC are smaller for -helical residues (J=9.6 ± 0.3 Hz) compared to -sheet (J=10.9 ± 0.8 Hz) and a substantial difference is observed for2JNC in -helices (J=6.4 ± 0.4 Hz) and -sheets (J=8.3 ± 0.8 Hz).Dedicated to the memory of Professor V.F. Bystrov 相似文献
19.
13C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T2 measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – 13C4-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B0. We have measured T2s, CSAs, and linewidths for the choline 13C--methyl, the cholesterol-C4 carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B0 field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and 13C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), 13C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined. 相似文献
20.
High resolution structural elucidation of macromolecular structure by solid-state nuclear magnetic resonance requires the preparation of uniformly aligned samples that are isotopically labeled. In addition, to use the chemical shift interaction as a high resolution constraint requires an in situ tensor characterization for each site of interest. For 13C in the peptide backbone, this characterization is complicated by the presence of dipolar coupled 14N from the peptide bond. Here the 13C1-Gly2 site in gramicidin A is studied both as a dry powder and in a fully hydrated lipid bilayer environment. Linewidths reported for the oriented samples are a factor of five narrower than those reported elsewhere, and previous misinterpretations of the linewidths are corrected. The observed frequency from oriented samples is shown to be consistent with the recently determined structure for this site in the gramicidin backbone. It is also shown that, whereas a dipolar coupling between 13C and 14N is apparent in dry preparations of the polypeptide, in a hydrated bilayer the dipolar coupling is absent, presumably due to a `self-decoupling' mechanism. 相似文献