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1.
A pulse sequence that yields three-dimensional 1H chemical shift / 1H-15N heteronuclear dipolar coupling / 15N chemical shift solid-state NMR spectra is demonstrated on a uniformly 15N labeled membrane protein in magnetically aligned phospholipid bilayers. Based on SAMPI4, the pulse sequence yields high
resolution in all three dimensions at a 1H resonance frequency of 900 MHz with the relatively low rf field strength (33 kHz) available for a lossy aqueous sample with
a commercial spectrometer and probe. The 1H chemical shift frequency dimension is shown to select among amide resonances, which will be useful in studies of larger
polytopic membrane proteins where the resonances overlap in two-dimensional spectra. Moreover, the 1H chemical shift, which can be measured from these spectra, provides an additional orientationally dependent frequency as
input for structure calculations.
Both Alexander A. Nevzorov and Sang Ho Park contributed equally to this work. 相似文献
2.
Evgeniy Salnikov Philippe Bertani Jan Raap Burkhard Bechinger 《Journal of biomolecular NMR》2009,45(4):373-387
In protein NMR spectroscopy the chemical shift provides important information for the assignment of residues and a first structural
evaluation of dihedral angles. Furthermore, angular restraints are obtained from oriented samples by solution and solid-state
NMR spectroscopic approaches. Whereas the anisotropy of chemical shifts, quadrupolar couplings and dipolar interactions have
been used to determine the structure, dynamics and topology of oriented membrane polypeptides using solid-state NMR spectroscopy
similar concepts have been introduced to solution NMR through the measurements of residual dipolar couplings. The analysis
of 15N chemical shift spectra depends on the accuracy of the chemical shift tensors. When investigating alamethicin and other peptaibols,
i.e. polypeptides rich in α-aminoisobutyric acid (Aib), the 15N chemical shift tensor of this Cα-tetrasubstituted amino acid exhibits pronounced differences when compared to glycine, alanine and other proteinogenic residues.
Here we present an experimental investigation on the 15N amide Aib tensor of N-acetyl-Aib-OH and for the Aib residues within peptaibols. Furthermore, a statistical analysis of the tensors published for
di- (glycine) and tri-substituted residues has been performed, where for the first time the published data sets are compiled
using a common reference. The size of the isotropic chemical shift and main tensor elements follows the order di- < tri- < tetra-substituted
amino acids. A 15N chemical shift-1H-15N dipolar coupling correlation NMR spectrum of alamethicin is used to evaluate the consequences of variations in the main
tensor elements for the structural analysis of this membrane peptide. 相似文献
3.
Nielsen NC Daugaard P Langer V Thomsen JK Nielsen S Sørensen OW Jakobsen HJ 《Journal of biomolecular NMR》1995,5(3):311-314
Summary A novel flat-coil solid-state NMR probe capable of controlling the hydration of oriented phospholipid bilayers in the course of long-term experiments, is described. Perfect hydration control for at least five days of intense radio-frequency pulsing is demonstrated using 31P NMR of oriented dimyristoylphospha-tidylcholine bilayers. The probe design will be of particular importance for studies of peptides and proteins oriented in lipid bilayers. 相似文献
4.
Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the ??shotgun?? approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the 15N chemical shift and 15N?C1H dipolar couplings, as well as spin-correlations between the (i, i?±?1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an 15N?C15N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled ??seed?? spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B 1 fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations. 相似文献
5.
F.M. Marassi J.J. Gesell A.P. Valente Y. Kim M. Oblatt-Montal M. Montal S.J. Opella 《Journal of biomolecular NMR》1999,14(2):141-148
The assignment of amide resonances in the two-dimensional PISEMA (Polarization Inversion with Spin Exchange at the Magic Angle) spectrum of uniformly 15N labeled M2 peptide corresponding to the channel-lining segment of the acetylcholine receptor in oriented phospholipid bilayers is described. The majority of the resonances were assigned through comparisons with spectra from selectively 15N labeled recombinant peptides and specifically 15N labeled synthetic peptides. Some resonances were assigned to specific amino acid residues by means of homonuclear 15N spin-exchange spectroscopy. A modification to the conventional spin-exchange pulse sequence that significantly shortens the length of the experiments by combining the intervals for 15 N spin-exchange and 1H magnetization recovery is described. 相似文献
6.
7.
Yasushi Oda Haruki Nakamura Shigenori Kanaya Morio Ikehara 《Journal of biomolecular NMR》1991,1(3):247-255
Summary The divalent metal ion binding site and binding constant of ribonuclease HI fromEscherichia coli were investigated by observing chemical shift changes using1H–15N heteronuclear NMR. Chemical shift changes were monitored during the titration of the enzyme with salts of the divalent cations. The enzyme was uniformly labeled by15N, which facilitated the monitoring of the chemical shift change of each cross peak between the backbone amide proton and the amide15N. The chemical shifts of several amide groups were affected upon the addition of a divalent metal ion: Mg2+, Ca2+, or Ba2+. These amide groups resided close to the active site, consistent with the previous X-ray crystallographic studies. From the titration analysis, a single divalent ion binding site was observed with a weak binding constant (KD=2–4 mM for the current divalent ions). 相似文献
8.
Shi P Xi Z Wang H Shi C Xiong Y Tian C 《Biochemical and biophysical research communications》2010,402(3):461-466
SH3 is a ubiquitous domain mediating protein-protein interactions. Recent solution NMR structural studies have shown that a proline-rich peptide is capable of binding to the human vinexin SH3 domain. Here, an orthogonal amber tRNA/tRNA synthetase pair for 15N/19F-trifluoromethyl-phenylalanine (15N/19F-tfmF) has been applied to achieve site-specific labeling of SH3 at three different sites. One-dimensional solution NMR spectra of backbone amide (15N)1H and side-chain 19F were obtained for SH3 with three different site-specific labels. Site-specific backbone amide (15N)1H and side-chain 19F chemical shift and relaxation analysis of SH3 in the absence or presence of a peptide ligand demonstrated different internal motions upon ligand binding at the three different sites. This site-specific NMR analysis might be very useful for studying large-sized proteins or protein complexes. 相似文献
9.
Orientation of cecropin A helices in phospholipid bilayers determined by solid-state NMR spectroscopy
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The orientation of the insect antibiotic peptide cecropin A (CecA) in the phospholipid bilayer membrane was determined using (15)N solid-state NMR spectroscopy. Two peptide samples, each specifically labeled with (15)N at Val(11) or Ala(27), were synthesized by solid phase techniques. The peptides were incorporated into phospholipid bilayers, prepared from a mixture of dimyristoylphosphatidylcholine and dimyristoylphosphatidylglycerol, and oriented on glass slides. The (15)N chemical shift solid-state NMR spectra from these uniaxially oriented samples display a single (15)N chemical shift frequency for each labeled residue. Both frequencies are near the upfield end of the (15)N chemical shift powder pattern, as expected for an alpha-helix with its long axis in the plane of the membrane and the NH bonds perpendicular to the direction of the magnetic field. These results support a mechanism of action in which CecA binds to and covers the membrane surface, thereby causing a general destabilization and leakiness of the lipid bilayer membrane. The data are discussed in relation to a proposed mechanism of membrane lysis and bacterial killing via an ion channel activity of CecA. 相似文献
10.
Jarbas M. Resende Rodrigo M. Verly Christopher Aisenbrey Amary Cesar Philippe Bertani Dorila Piló-Veloso Burkhard Bechinger 《Biophysical journal》2014
Phylloseptin-1, -2, and -3 are three members of the family of linear cationic antimicrobial peptides found in tree frogs. The highly homologous peptides encompass 19 amino acids, and only differ in the amino acid composition and charge at the six most carboxy-terminal residues. Here, we investigated how such subtle changes are reflected in their membrane interactions and how these can be correlated to their biological activities. To this end, the three peptides were labeled with stable isotopes, reconstituted into oriented phospholipid bilayers, and their detailed topology determined by a combined approach using 2H and 15N solid-state NMR spectroscopy. Although phylloseptin-2 and -3 adopt perfect in-plane alignments, the tilt angle of phylloseptin-1 deviates by 8° probably to assure a more water exposed localization of the lysine-17 side chain. Furthermore, different azimuthal angles are observed, positioning the amphipathic helices of all three peptides with the charged residues well exposed to the water phase. Interestingly, our studies also reveal that two orientation-dependent 2H quadrupolar splittings from methyl-deuterated alanines and one 15N amide chemical shift are sufficient to unambiguously determine the topology of phylloseptin-1, where quadrupolar splittings close to the maximum impose the most stringent angular restraints. As a result of these studies, a strategy is proposed where the topology of a peptide structure can be determined accurately from the labeling with 15N and 2H isotopes of only a few amino acid residues. 相似文献
11.
Jarbas?M. Resende Rodrigo?M. Verly Christopher Aisenbrey Amary Cesar Philippe Bertani Dorila Piló-Veloso Burkhard Bechinger 《Biophysical journal》2014,107(4):901-911
Phylloseptin-1, -2, and -3 are three members of the family of linear cationic antimicrobial peptides found in tree frogs. The highly homologous peptides encompass 19 amino acids, and only differ in the amino acid composition and charge at the six most carboxy-terminal residues. Here, we investigated how such subtle changes are reflected in their membrane interactions and how these can be correlated to their biological activities. To this end, the three peptides were labeled with stable isotopes, reconstituted into oriented phospholipid bilayers, and their detailed topology determined by a combined approach using 2H and 15N solid-state NMR spectroscopy. Although phylloseptin-2 and -3 adopt perfect in-plane alignments, the tilt angle of phylloseptin-1 deviates by 8° probably to assure a more water exposed localization of the lysine-17 side chain. Furthermore, different azimuthal angles are observed, positioning the amphipathic helices of all three peptides with the charged residues well exposed to the water phase. Interestingly, our studies also reveal that two orientation-dependent 2H quadrupolar splittings from methyl-deuterated alanines and one 15N amide chemical shift are sufficient to unambiguously determine the topology of phylloseptin-1, where quadrupolar splittings close to the maximum impose the most stringent angular restraints. As a result of these studies, a strategy is proposed where the topology of a peptide structure can be determined accurately from the labeling with 15N and 2H isotopes of only a few amino acid residues. 相似文献
12.
15N- and 2H-substituted maleimido-TEMPO spin label ([15N,2H]MTSL) and the fluorescent label 1,5-IAEDANS were used to specifically modify sulfhydryl 1 of myosin to study the orientation of myosin cross-bridges in skeletal muscle fibers. The electron paramagnetic resonance (EPR) spectrum from muscle fibers decorated with labeled myosin subfragment 1 ([15N,2H]MTSL-S1) or the fluorescence polarization spectrum from fibers directly labeled with 1,5-IAEDANS was measured from fibers in various physiological conditions. The EPR spectra from fibers with the fiber axis oriented at 90 degrees to the Zeeman field show a clear spectral shift from the rigor spectrum when the myosin cross-bridge binds MgADP. This shift is attributable to a change in the torsion angle of the spin probe from cross-bridge rotation and is observable due mainly to the improved angular resolution of the substituted probe. The EPR data from [15N,2H]MTSL-S1 decorating fibers are combined with the fluorescence polarization data from the 1,5-IAEDANS-labeled fibers to map the global angular transition of the labeled cross-bridges due to nucleotide binding by an analytical method described in the accompanying paper [Burghardt, T. P., & Ajtai, K. (1992) Biochemistry (preceding paper in this issue)]. We find that the spin and fluorescent probes are quantitatively consistent in the finding that the actin-bound cross-bridge rotates through a large angle upon binding MgADP. We also find that, if the shape of the cross-bridge is described as an ellipsoid with two equivalent minor axes, then cross-bridge rotation takes place mainly about an axis parallel to the major axis of the ellipsoid. This type of rotation may imitate the rotation motion of cross-bridges during force generation. 相似文献
13.
Maureen Leninger William M. Marsiglia Alexej Jerschow Nathaniel J. Traaseth 《Journal of biomolecular NMR》2018,71(1):19-30
Exchange between conformational states is required for biomolecular catalysis, allostery, and folding. A variety of NMR experiments have been developed to quantify motional regimes ranging from nanoseconds to seconds. In this work, we describe an approach to speed up the acquisition of chemical exchange saturation transfer (CEST) experiments that are commonly used to probe millisecond to second conformational exchange in proteins and nucleic acids. The standard approach is to obtain CEST datasets through the acquisition of a series of 2D correlation spectra where each experiment utilizes a single saturation frequency to 1H, 15N or 13C. These pseudo 3D datasets are time consuming to collect and are further lengthened by reduced signal to noise stemming from the long saturation pulse. In this article, we show how usage of a multiple frequency saturation pulse (i.e., MF-CEST) changes the nature of data collection from series to parallel, and thus decreases the total acquisition time by an integer factor corresponding to the number of frequencies in the pulse. We demonstrate the applicability of MF-CEST on a Src homology 2 (SH2) domain from phospholipase Cγ and the secondary active transport protein EmrE as model systems by collecting 13C methyl and 15N backbone datasets. MF-CEST can also be extended to additional sites within proteins and nucleic acids. The only notable drawback of MF-CEST as applied to backbone 15N experiments occurs when a large chemical shift difference between the major and minor populations is present (typically greater than ~?8 ppm). In these cases, ambiguity may arise between the chemical shift of the minor population and the multiple frequency saturation pulse. Nevertheless, this drawback does not occur for methyl group MF-CEST experiments or in cases where somewhat smaller chemical shift differences occur are present. 相似文献
14.
Mark P. Foster Deborah S. Wuttke Karen R. Clemens Wolfgang Jahnke Ishwar Radhakrishnan Linda Tennant Martine Reymond John Chung Peter E. Wright 《Journal of biomolecular NMR》1998,12(1):51-71
We report the NMR resonance assignments for a macromolecular protein/DNA complex containing the three amino-terminal zinc fingers (92 amino acid residues) of Xenopus laevis TFIIIA (termed zf1-3) bound to the physiological DNA target (15 base pairs), and for the free DNA. Comparisons are made of the chemical shifts of protein backbone1 HN, 15N,13 C and13 C and DNA base and sugar protons of the free and bound species. Chemical shift changes are analyzed in the context of the structures of the zf1-3/DNA complex to assess the utility of chemical shift change as a probe of molecular interfaces. Chemical shift perturbations that occur upon binding in the zf1-3/DNA complex do not correspond directly to the structural interface, but rather arise from a number of direct and indirect structural and dynamic effects. 相似文献
15.
Rodrigo M. Verly Jarbas M. Resende Christopher Aisenbrey Dorila Piló-Veloso Fábio C.L. Almeida 《Biophysical journal》2009,96(6):2194-2203
DD K, a peptide first isolated from the skin secretion of the Phyllomedusa distincta frog, has been prepared by solid-phase chemical peptide synthesis and its conformation was studied in trifluoroethanol/water as well as in the presence of sodium dodecyl sulfate and dodecylphosphocholine micelles or small unilamellar vesicles. Multidimensional solution NMR spectroscopy indicates an α-helical conformation in membrane environments starting at residue 7 and extending to the C-terminal carboxyamide. Furthermore, DD K has been labeled with 15N at a single alanine position that is located within the helical core region of the sequence. When reconstituted into oriented phosphatidylcholine membranes the resulting 15N solid-state NMR spectrum shows a well-defined helix alignment parallel to the membrane surface in excellent agreement with the amphipathic character of DD K. Proton-decoupled 31P solid-state NMR spectroscopy indicates that the peptide creates a high level of disorder at the level of the phospholipid headgroup suggesting that DD K partitions into the bilayer where it severely disrupts membrane packing. 相似文献
16.
Leppert J Heise B Ohlenschläger O Görlach M Ramachandran R 《Journal of biomolecular NMR》2004,28(2):185-190
A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly (13C, 15N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear 13C-15N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power 1H decoupling in experiments requiring long t
1 acquisition times. 相似文献
17.
Robert Tycko 《Journal of biomolecular NMR》1996,8(3):239-251
Summary The feasibility of assigning the backbone 15N and 13C NMR chemical shifts in multidimensional magic angle spinning NMR spectra of uniformly isotopically labeled proteins and peptides in unoriented solid samples is assessed by means of numerical simulations. The goal of these simulations is to examine how the upper limit on the size of a peptide for which unique assignments can be made depends on the spectral resolution, i.e., the NMR line widths. Sets of simulated three-dimensional chemical shift correlation spectra for artificial peptides of varying length are constructed from published liquid-state NMR chemical shift data for ubiquitin, a well-characterized soluble protein. Resonance assignments consistent with these spectra to within the assumed spectral resolution are found by a numerical search algorithm. The dependence of the number of consistent assignments on the assumed spectral resolution and on the length of the peptide is reported. If only three-dimensional chemical shift correlation data for backbone 15N and 13C nuclei are used, and no residue-specific chemical shift information, information from amino acid side-chain signals, and proton chemical shift information are available, a spectral resolution of 1 ppm or less is generally required for a unique assignment of backbone chemical shifts for a peptide of 30 amino acid residues. 相似文献
18.
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
19.
Chemical shift assignment is reported for the protein ubiquitin denatured in 8M urea at pH 2. The variations in 15N chemical shifts of three different proteins (ubiquitin, disulfide reduced, carboxymethylated lysozyme, all-Ala--lactalbumin), all without disulfides and denatured in 8M urea at pH 2 are compared to `random coil shifts' of small model peptides (Braun et al., 1994) and to the averaged native chemical shifts taken from the BMRB database. Both parameterizations show a remarkable agreement with the averaged measured 15N chemical shifts in the three denatured proteins. Detailed analysis of these experimental 15N chemical shifts provides an estimate of the influence of nearest neighbors and conformational preferences on the chemical shift and provides a direct means to identify non-random structural preferences in denatured proteins. 相似文献
20.
Salnikov ES Friedrich H Li X Bertani P Reissmann S Hertweck C O'Neil JD Raap J Bechinger B 《Biophysical journal》2009,96(1):86-100
Ampullosporin A and alamethicin are two members of the peptaibol family of antimicrobial peptides. These compounds are produced by fungi and are characterized by a high content of hydrophobic amino acids, and in particular the α-tetrasubstituted amino acid residue α-aminoisobutyric acid. Here ampullosporin A and alamethicin were uniformly labeled with 15N, purified and reconstituted into oriented phophatidylcholine lipid bilayers and investigated by proton-decoupled 15N and 31P solid-state NMR spectroscopy. Whereas alamethicin (20 amino acid residues) adopts transmembrane alignments in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes the much shorter ampullosporin A (15 residues) exhibits comparable configurations only in thin membranes. In contrast the latter compound is oriented parallel to the membrane surface in 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine and POPC bilayers indicating that hydrophobic mismatch has a decisive effect on the membrane topology of these peptides. Two-dimensional 15N chemical shift - 1H-15N dipolar coupling solid-state NMR correlation spectroscopy suggests that in their transmembrane configuration both peptides adopt mixed α-/310-helical structures which can be explained by the restraints imposed by the membranes and the bulky α-aminoisobutyric acid residues. The 15N solid-state NMR spectra also provide detailed information on the helical tilt angles. The results are discussed with regard to the antimicrobial activities of the peptides. 相似文献