共查询到20条相似文献,搜索用时 31 毫秒
1.
Heinzmann R Grage SL Schalck C Bürck J Bánóczi Z Toke O Ulrich AS 《European biophysics journal : EBJ》2011,40(4):463-470
The preceding contribution by Toke et al. has studied the structure of the cationic antimicrobial peptide maximin-4 in detergent
micelles and in organic solvent, revealing a different kink angle and side-chain interactions in the two different environments.
Here, we have examined the same peptide in lipid bilayers using oriented circular dichroism (OCD) and solid-state 15N nuclear magnetic resonance (NMR) in aligned samples. OCD showed that maximin-4 is helical and adopts an oblique alignment
in the membrane, and lacks the characteristic realignment response that is often observed for amphipathic α-helical peptides
at a peptide:lipid ratio between 1:100 and 1:20. Solid-state 15N-NMR experiments suggest that maximin-4 also remains unaffected by lipid charge and temperature. Analyzing 15N labels in positions Ala12, Ala13, and Leu14, an oblique tilt angle of the N-terminal helix of ~130° relative to the membrane
normal was found, in good agreement with the amphiphilic profile of this segment. An additional constraint at Ala22 in the
C-terminal segment is found to be compatible with a continuous α-helix, but unfavorable side-chain interactions make this
solution unlikely. Instead, a kink at Gly16 seems fully compatible with all known constraints and with the biophysical expectations
in the membrane-bound state, given the liquid-state NMR structures. It thus seems that the flexible kink in maximin-4 allows
the two helical segments to adjust to the local environment. The irregular amphiphilic profile and the resulting versatility
in shape might explain why maximin-4 lacks the realignment response that has been characteristically observed for many related
frog peptides forming straight amphipathic α-helices. 相似文献
2.
Julia?Misiewicz Sergii?Afonin Stephan?L.?Grage Jonas?van den?Berg Erik?Strandberg Parvesh?Wadhwani Anne?S.?Ulrich
Membrane composition is a key factor that regulates the destructive activity of antimicrobial peptides and the non-leaky permeation of cell penetrating peptides in vivo. Hence, the choice of model membrane is a crucial aspect in NMR studies and should reflect the biological situation as closely as possible. Here, we explore the structure and dynamics of the short multifunctional peptide BP100 using a multinuclear solid-state NMR approach. The membrane alignment and mobility of this 11 amino acid peptide was studied in various synthetic lipid bilayers with different net charge, fluidity, and thickness, as well as in native biomembranes harvested from prokaryotic and eukaryotic cells. 19F-NMR provided the high sensitivity and lack of natural abundance background that are necessary to observe a labelled peptide even in protoplast membranes from Micrococcus luteus and in erythrocyte ghosts. Six selectively 19F-labeled BP100 analogues gave remarkably similar spectra in all of the macroscopically oriented membrane systems, which were studied under quasi-native conditions of ambient temperature and full hydration. This similarity suggests that BP100 has the same surface-bound helical structure and high mobility in the different biomembranes and model membranes alike, independent of charge, thickness or cholesterol content of the system. 31P-NMR spectra of the phospholipid components did not indicate any bilayer perturbation, so the formation of toroidal wormholes or micellarization can be excluded as a mechanism of its antimicrobial or cell penetrating action. However, 2H-NMR analysis of the acyl chain order parameter profiles showed that BP100 leads to considerable membrane thinning and thereby local destabilization. 相似文献
3.
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
4.
Grasnick D Sternberg U Strandberg E Wadhwani P Ulrich AS 《European biophysics journal : EBJ》2011,40(4):529-543
To better understand peptide-induced membrane fusion at a molecular level, we set out to determine the structure of the fusogenic
peptide FP23 from the HIV-1 protein gp41 when bound to a lipid bilayer. An established solid-state 19F nuclear magnetic resonance (NMR) approach was used to collect local orientational constraints from a series of CF3-phenylglycine-labeled peptide analogues in macroscopically aligned membranes. Fusion assays showed that these 19F-labels did not significantly affect peptide function. The NMR spectra were characteristic of well-behaved samples, without
any signs of heterogeneity or peptide aggregation at 1:300 in 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). We can conclude from these NMR data that FP23 has a well-defined (time-averaged) conformation
and undergoes lateral diffusion in the bilayer plane, presumably as a monomer or small oligomer. Attempts to evaluate its
conformation in terms of various secondary structures, however, showed that FP23 does not form any type of regular helix or
β-strand. Therefore, all-atom molecular dynamics (MD) simulations were carried out using the orientational NMR constraints
as pseudo-forces to drive the peptide into a stable alignment and structure. The resulting picture suggests that FP23 can
adopt multiple β-turns and insert obliquely into the membrane. Such irregular conformation explains why the structure of the
fusion peptide could not be reliably determined by any biophysical method so far. 相似文献
5.
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
6.
Mariana C. Manzini Katia R. Perez Karin A. Riske José C. Bozelli Jr. Talita L. Santos Marcia A. da Silva Greice K.V. Saraiva Mario J. Politi Ana P. Valente Fábio C.L. Almeida Hernan Chaimovich Magali A. Rodrigues Marcelo P. Bemquerer Shirley Schreier Iolanda M. Cuccovia 《生物化学与生物物理学报:生物膜》2014
The cecropin-melittin hybrid antimicrobial peptide BP100 (H-KKLFKKILKYL-NH2) is selective for Gram-negative bacteria, negatively charged membranes, and weakly hemolytic. We studied BP100 conformational and functional properties upon interaction with large unilamellar vesicles, LUVs, and giant unilamellar vesicles, GUVs, containing variable proportions of phosphatidylcholine (PC) and negatively charged phosphatidylglycerol (PG). CD and NMR spectra showed that upon binding to PG-containing LUVs BP100 acquires α-helical conformation, the helix spanning residues 3–11. Theoretical analyses indicated that the helix is amphipathic and surface-seeking. CD and dynamic light scattering data evinced peptide and/or vesicle aggregation, modulated by peptide:lipid ratio and PG content. BP100 decreased the absolute value of the zeta potential (ζ) of LUVs with low PG contents; for higher PG, binding was analyzed as an ion-exchange process. At high salt, BP100-induced LUVS leakage requires higher peptide concentration, indicating that both electrostatic and hydrophobic interactions contribute to peptide binding. While a gradual release took place at low peptide:lipid ratios, instantaneous loss occurred at high ratios, suggesting vesicle disruption. Optical microscopy of GUVs confirmed BP100-promoted disruption of negatively charged membranes. The mechanism of action of BP100 is determined by both peptide:lipid ratio and negatively charged lipid content. While gradual release results from membrane perturbation by a small number of peptide molecules giving rise to changes in acyl chain packing, lipid clustering (leading to membrane defects), and/or membrane thinning, membrane disruption results from a sequence of events – large-scale peptide and lipid clustering, giving rise to peptide-lipid patches that eventually would leave the membrane in a carpet-like mechanism. 相似文献
7.
Matthew J. McKay Ashley N. Martfeld Anna A. De Angelis Stanley J. Opella Denise V. Greathouse Roger E. Koeppe 《Biophysical journal》2018,114(11):2617-2629
Transmembrane protein domains often contain interfacial aromatic residues, which may play a role in the insertion and stability of membrane helices. Residues such as Trp or Tyr, therefore, are often found situated at the lipid-water interface. We have examined the extent to which the precise radial locations of interfacial Trp residues may influence peptide helix orientation and dynamics. To address these questions, we have modified the GW5,19ALP23 (acetyl-GGALW5(LA)6LW19LAGA-[ethanol]amide) model peptide framework to relocate the Trp residues. Peptide orientation and dynamics were analyzed by means of solid-state nuclear magnetic resonance (NMR) spectroscopy to monitor specific 2H- and 15N-labeled residues. GW5,19ALP23 adopts a defined, tilted orientation within lipid bilayer membranes with minimal evidence of motional averaging of NMR observables, such as 2H quadrupolar or 15N-1H dipolar splittings. Here, we examine how peptide dynamics are impacted by relocating the interfacial Trp (W) residues on both ends and opposing faces of the helix, for example by a 100° rotation on the helical wheel for positions 4 and 20. In contrast to GW5,19ALP23, the modified GW4,20ALP23 helix experiences more extensive motional averaging of the NMR observables in several lipid bilayers of different thickness. Individual and combined Gaussian analyses of the 2H and 15N NMR signals confirm that the extent of dynamic averaging, particularly rotational “slippage” about the helix axis, is strongly coupled to the radial distribution of the interfacial Trp residues as well as the bilayer thickness. Additional 2H labels on alanines A3 and A21 reveal partial fraying of the helix ends. Even within the context of partial unwinding, the locations of particular Trp residues around the helix axis are prominent factors for determining transmembrane helix orientation and dynamics within the lipid membrane environment. 相似文献
8.
Grage SL Strandberg E Wadhwani P Esteban-Martín S Salgado J Ulrich AS 《European biophysics journal : EBJ》2012,41(5):475-482
Many solid-state nuclear magnetic resonance (NMR) approaches for membrane proteins rely on orientation-dependent parameters,
from which the alignment of peptide segments in the lipid bilayer can be calculated. Molecules embedded in liquid-crystalline
membranes, such as monomeric helices, are highly mobile, leading to partial averaging of the measured NMR parameters. These
dynamic effects need to be taken into account to avoid misinterpretation of NMR data. Here, we compare two common NMR approaches:
2H-NMR quadrupolar waves, and separated local field 15N–1H polarization inversion spin exchange at magic angle (PISEMA) spectra, in order to identify their strengths and drawbacks
for correctly determining the orientation and mobility of α-helical transmembrane peptides. We first analyzed the model peptide
WLP23 in oriented dimyristoylphosphatidylcholine (DMPC) membranes and then contrasted it with published data on GWALP23 in
dilauroylphosphatidylcholine (DLPC). We only obtained consistent tilt angles from the two methods when taking dynamics into
account. Interestingly, the two related peptides differ fundamentally in their mobility. Although both helices adopt the same
tilt in their respective bilayers (~20°), WLP23 undergoes extensive fluctuations in its azimuthal rotation angle, whereas
GWALP23 is much less dynamic. Both alternative NMR methods are suitable for characterizing orientation and dynamics, yet they
can be optimally used to address different aspects. PISEMA spectra immediately reveal the presence of large-amplitude rotational
fluctuations, which are not directly seen by 2H-NMR. On the other hand, PISEMA was unable to define the azimuthal rotation angle in the case of the highly dynamic WLP23,
though the helix tilt could still be determined, irrespective of any dynamics parameters. 相似文献
9.
Lipopeptide MSI-843 consisting of the nonstandard amino acid ornithine (Oct-OOLLOOLOOL-NH2) was designed with an objective towards generating non-lytic short antimicrobial peptides, which can have significant pharmaceutical applications. Octanoic acid was coupled to the N-terminus of the peptide to increase the overall hydrophobicity of the peptide. MSI-843 shows activity against bacteria and fungi at micromolar concentrations. It permeabilizes the outer membrane of Gram-negative bacterium and a model membrane mimicking bacterial inner membrane. Circular dichroism investigations demonstrate that the peptide adopts α-helical conformation upon binding to lipid membranes. Isothermal titration calorimetry studies suggest that the peptide binding to membranes results in exothermic heat of reaction, which arises from helix formation and membrane insertion of the peptide. 2H NMR of deuterated-POPC multilamellar vesicles shows the peptide-induced disorder in the hydrophobic core of bilayers. 31P NMR data indicate changes in the lipid head group orientation of POPC, POPG and Escherichia colitotal lipid bilayers upon peptide binding. Results from 31P NMR and dye leakage experiments suggest that the peptide selectively interacts with anionic bilayers at low concentrations (up to 5 mol%). Differential scanning calorimetry experiments on DiPOPE bilayers and 31P NMR data from E.coli total lipid multilamellar vesicles indicate that MSI-843 increases the fluid lamellar to inverted hexagonal phase transition temperature of bilayers by inducing positive curvature strain. Combination of all these data suggests the formation of a lipid-peptide complex resulting in a transient pore as a plausible mechanism for the membrane permeabilization and antimicrobial activity of the lipopeptide MSI-843. 相似文献
10.
Solution Synthesis,Conformational Analysis,and Antimicrobial Activity of Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label
下载免费PDF全文
![点击此处可从《化学与生物多样性》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Marta De Zotti Gema Ballano Micha Jost Evgeniy S. Salnikov Burkhard Bechinger Simona Oancea Marco Crisma Claudio Toniolo Fernando Formaggio 《化学与生物多样性》2014,11(8):1163-1191
We prepared, by solution‐phase methods, and fully characterized three analogs of the membrane‐active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N‐terminus, at position 9 (central region) or at position 19 (C‐terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ‐trifluoroacetylated α,γ‐diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val9 or Glu(OMe)19 amino acid. We performed a detailed conformational analysis of the three analogs (using FT‐IR absorption, CD, 2D‐NMR, and X‐ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α‐helical structure of the parent peptaibiotic. The results of an initial solid‐state 19F‐NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibiotic? membrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence. 相似文献
11.
12.
Diana E. Wetzler Mariana Gallo Riccardo Melis Tomasso Eliseo Alejandro D. Nadra Diego U. Ferreiro Maurizio Paci Ignacio E. Sánchez Daniel O. Cicero Gonzalo de Prat Gay 《Biopolymers》2009,91(6):432-443
Nucleic acid recognition is often mediated by α‐helices or disordered regions that fold into α‐helix on binding. A peptide bearing the DNA recognition helix of HPV16 E2 displays type II polyproline (PII) structure as judged by pH, temperature, and solvent effects on the CD spectra. NMR experiments indicate that the canonical α‐helix is stabilized at the N‐terminus, while the PII forms at the C‐terminus half of the peptide. Re‐examination of the dihedral angles of the DNA binding helix in the crystal structure and analysis of the NMR chemical shift indexes confirm that the N‐terminus half is a canonical α‐helix, while the C‐terminal half adopts a 310 helix structure. These regions precisely match two locally driven folding nucleii, which partake in the native hydrophobic core and modulate a conformational switch in the DNA binding helix. The peptide shows only weak and unspecific residual DNA binding, 104‐fold lower affinity, and 500‐fold lower discrimination capacity compared with the domain. Thus, the precise side chain conformation required for modulated and tight physiological binding by HPV E2 is largely determined by the noncanonical strained α‐helix conformation, “presented” by this unique architecture. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 432–443, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com 相似文献
13.
Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR 总被引:1,自引:0,他引:1
下载免费PDF全文
![点击此处可从《Biophysical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Glaser RW Sachse C Dürr UH Wadhwani P Afonin S Strandberg E Ulrich AS 《Biophysical journal》2005,88(5):3392-3397
The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration. 相似文献
14.
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
15.
Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism
Bürck J Roth S Wadhwani P Afonin S Kanithasen N Strandberg E Ulrich AS 《Biophysical journal》2008,95(8):3872-3881
Oriented circular dichroism (OCD) was used to characterize and compare in a quantitative manner the secondary structure and concentration dependent realignment of the antimicrobial peptides PGLa and MSI-103, and of the structurally related cell-penetrating peptide MAP in aligned phospholipid bilayers. All these peptides adopt an amphiphilic α-helical conformation, and from solid-state NMR analysis they are known to bind to membranes in two distinct orientations depending on their concentration. At low peptide/lipid (P/L) ratio the helices are aligned parallel to membrane surface (S-state), but with increasing concentration they realign to a tilted orientation (T-state), getting immersed into the membrane with an oblique angle supposedly as a result of dimer-formation. In macroscopically aligned liquid crystalline 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers the two limiting states are represented by distinct OCD spectra, and all spectra at intermediate peptide concentrations can be described by a linear combination of these two line shapes. The corresponding fraction of molecules occupying the T-state was determined by fitting the intermediate spectra with a superposition of the two extreme line shapes. By plotting this fraction versus 1/(P/L), the threshold P/L* ratio for realignment was extracted for each of the three related peptides. Despite their structural similarity distinctly different thresholds were obtained, namely for MSI-103 realignment starts already at a low P/L of ∼1:236, for a MAP derivative (using a nonaggregating analog containing a D-amino acid) the transition begins at P/L ∼1:156, whereas PGLa needs the highest concentration to flip into T-state at P/L ∼1:85. Analysis of the original MAP sequence (containing only L-amino acids) gave OCD spectra compatible with β-pleated conformation, suggesting that this peptide starts to aggregate with increasing concentration, unlike the other helical peptides. All these changes in peptide conformation and membrane alignment observed here by OCD seem to be functionally relevant, as they can be correlated with the membrane perturbing activities of the three antimicrobial and cell-penetrating sequences. 相似文献
16.
Burkhard Bechinger 《Proteins》1997,27(4):481-492
The polypeptide corresponding to the signal sequence of the M13 coat protein and the five N-terminal residues of the mature protein was prepared by solid-phase peptide synthesis with a 15N isotopic label at the alanine-12 position. Multidimensional solution NMR spectroscopy and molecular modeling calculations indicate that this polypeptide assumes helical conformations between residues 5 and 20, in the presence of sodium dodecylsulfate micelles. This is in good agreement with circular dichroism spectroscopic measurement, which shows an α-helix content of approximately 42%. The α-helix comprises an uninterrupted hydrophobic stretch of ≤12 amino acids, which is generally believed to be too short for a stable transmembrane alignment in a biological bilayer. The monoexponential proton-deuterium exchange kinetics of this hydrophobic helical region is characterized by half-lives of 15–75 minutes (pH 4.2, 323 K). When the polypeptide is reconstituted into phospholipid bilayers, the broad anisotropy of the proton-decoupled 15N solid-state NMR spectroscopy indicates that the hydrophobic helix is immobilized close to the lipid bilayer surface at the time scale of 15N solid-state NMR spectroscopy (10−4 seconds). By contrast, short correlation times, immediate hydrogen-deuterium exchange as well as nuclear Overhauser effect crosspeak analysis suggest that the N and C termini of this polypeptide exhibit a mobile random coil structure. The implications of these structural findings for possible mechanisms of membrane insertion and translocation as well as for membrane protein structure prediction algorithms are discussed. © 1997 Wiley-Liss Inc. 相似文献
17.
Aisenbrey C Bertani P Henklein P Bechinger B 《European biophysics journal : EBJ》2007,36(4-5):451-460
Knowledge of the structure, dynamics and interactions of polypeptides when associated with phospholipid bilayers is key to
understanding the functional mechanisms of channels, antibiotics, signal- or translocation peptides. Solid-state NMR spectroscopy
on samples uniaxially aligned relative to the magnetic field direction offers means to determine the alignment of polypeptide
bonds and domains relative to the bilayer normal. Using this approach the 15N chemical shift of amide bonds provides a direct indicator of the approximate helical tilt, whereas the 2H solid-state NMR spectra acquired from peptides labelled with 3,3,3-2H3-alanines contain valuable complimentary information for a more accurate analysis of tilt and rotation pitch angles. The deuterium
NMR line shapes are highly sensitive to small variations in the alignment of the Cα–Cβ bond relative to the magnetic field direction and, therefore, also the orientational distribution of helices relative to
the membrane normal. When the oriented membrane samples are investigated with their normal perpendicular to the magnetic field
direction, the rate of rotational diffusion can be determined in a semi-quantitative manner and thereby the aggregation state
of the peptides can be analysed. Here the deuterium NMR approach is first introduced showing results from model amphipathic
helices. Thereafter investigations of the viral channel peptides Vpu1–27 and Influenza A M222–46 are shown. Whereas the 15N chemical shift data confirm the transmembrane helix alignments of these hydrophobic sequences, the deuterium spectra indicate
considerable mosaic spread in the helix orientations. At least two peptide populations with differing rotational correlation
times are apparent in the deuterium spectra of the viral channels suggesting an equilibrium between monomeric peptides and
oligomeric channel configurations under conditions where solid-state NMR structural studies of these peptides have previously
been performed.
Dedicated to Prof. K. Arnold on the occasion of his 65th birthday. 相似文献
18.
《生物化学与生物物理学报:生物膜》2018,1860(2):407-415
The lethal Coronaviruses (CoVs), Severe Acute Respiratory Syndrome-associated Coronavirus (SARS-CoV) and most recently Middle East Respiratory Syndrome Coronavirus, (MERS-CoV) are serious human health hazard. A successful viral infection requires fusion between virus and host cells carried out by the surface spike glycoprotein or S protein of CoV. Current models propose that the S2 subunit of S protein assembled into a hexameric helical bundle exposing hydrophobic fusogenic peptides or fusion peptides (FPs) for membrane insertion. The N-terminus of S2 subunit of SARS-CoV reported to be active in cell fusion whereby FPs have been identified. Atomic-resolution structure of FPs derived either in model membranes or in membrane mimic environment would glean insights toward viral cell fusion mechanism. Here, we have solved 3D structure, dynamics and micelle localization of a 64-residue long fusion peptide or LFP in DPC detergent micelles by NMR methods. Micelle bound structure of LFP is elucidated by the presence of discretely folded helical and intervening loops. The C-terminus region, residues F42-Y62, displays a long hydrophobic helix, whereas the N-terminus is defined by a short amphipathic helix, residues R4-Q12. The intervening residues of LFP assume stretches of loops and helical turns. The N-terminal helix is sustained by close aromatic and aliphatic sidechain packing interactions at the non-polar face. 15N{1H}NOE studies indicated dynamical motion, at ps-ns timescale, of the helices of LFP in DPC micelles. PRE NMR showed that insertion of several regions of LFP into DPC micelle core. Together, the current study provides insights toward fusion mechanism of SARS-CoV. 相似文献
19.
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 CD3- or CF3-group attached to the CαCβ 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 15N-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. 相似文献
20.
Oriented solid-state NMR in combination with multiple-residue-specific 15N labeling and extensive numerical spectral analysis is proposed to determine helix conformations of large membrane proteins in native membranes. The method is demonstrated on uniaxially oriented samples of 15N-methionine, -valine, and -glycine-labeled bacteriorhopsin in native purple membranes. Experimental two-dimensional 1H-15N dipole-dipole coupling versus 15N chemical shift spectra for all samples are analyzed numerically to establish combined constraints on the orientation of the seven transmembrane helices relative to the membrane bilayer normal. Since the method does not depend on specific resonance assignments and proves robust toward nonidealities in the sample alignment, it may be generally feasible for the study of conformational arrangement and function-induced conformation changes of large integral membrane proteins. 相似文献