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1.
The dynamics of membrane-spanning peptides have a strong affect on the solid-state NMR observables. We present a combined analysis of 2H-alanine quadrupolar splittings together with 15N/1H dipolar couplings and 15N chemical shifts, using two models to treat the dynamics, for the systematic evaluation of transmembrane peptides based on the GWALP23 sequence (acetyl-GGALW(LA)6LWLAGA-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 2H- or 15N-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 15N as well as 2H 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 2H or 15N 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. 相似文献
2.
Aldo Franco Frank Marí 《International journal of peptide research and therapeutics》1999,6(4):199-207
Summary α-conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom ofConus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor
(nAChR) found in mammalian skeletal muscle and the electric organTorpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR
methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce
13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical
loop between THr9 and Met12 that is stabilized by the Cys4-Cys10 disulfide bond and turns involving Cys4-Cys5 and Asn14-Pro15. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B,
MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity
can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding
of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI.
Supplementary material available:1H resonance assignments of α-conotoxin EI. 相似文献
3.
Franco Aldo Marí Frank 《International journal of peptide research and therapeutics》1999,6(4):199-208
α-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other α-conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr9 and Met12 that is stabilized by the Cys4-Cys10 disulfide bond and turns involving Cys4-Cys5 and Asn14-Pro15. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other α4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other α4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these α4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI. 相似文献
4.
We applied precise densimetry and ultrasound velocimetry methods to study the interaction of a synthetic α-helical transmembrane peptide, acetyl-K2-L24-K2-amide (L24), with model bilayer lipid membranes. The large unilamellar vesicles (LUVs) utilized were composed of a homologous series of n-saturated diacylphosphatidylcholines (PCs). PCs whose hydrocarbon chains contained from 13 to 16 carbon atoms, thus producing phospholipid bilayers of different thicknesses and gel to liquid-crystalline phase transition temperatures. This allowed us to analyze how the difference between the hydrophobic length of the peptide and the hydrophobic thickness of the lipid bilayer influences the thermodynamical and mechanical properties of the membranes. We showed that the incorporation of L24 decreases the temperature and cooperativity of the main phase transition of all LUVs studied. The presence of L24 in the bilayer also caused an increase of the specific volume and of the volume compressibility in the gel state bilayers. In the liquid crystalline state, the peptide decreases the specific volume at relatively higher peptide concentration (mole ratio L24:PC = 1:50). The overall volume compressibility of the peptide-containing lipid bilayers in the liquid-crystalline state was in general higher in comparison with pure membranes. There was, however, a tendency for the volume compressibility of these lipid bilayers to decrease with higher peptide content in comparison with bilayers of lower peptide concentration. For one lipid composition, we also compared the thermodynamical and mechanical properties of LUVs and large multilamellar vesicles (MLVs) with and without L24. As expected, a higher cooperativity of the changes of the thermodynamical and mechanical parameters took place for MLVs in comparison with LUVs. These results are in agreement with previously reported DSC and 2H NMR spectroscopy study of the interaction of the L24 and structurally related peptides with phosphatidylcholine bilayers. An apparent discrepancy between 2H NMR spectroscopy and compressibility data in the liquid crystalline state may be connected with the complex and anisotropic nature of macroscopic mechanical properties of the membranes. The observed changes in membrane mechanical properties induced by the presence of L24 suggest that around each peptide a distorted region exists that involves at least 2 layers of lipid molecules. 相似文献
5.
Himanshu Khandelia 《生物化学与生物物理学报:生物膜》2007,1768(3):509-520
All atom molecular dynamics simulations of the 18-residue β-hairpin antimicrobial peptide protegrin-1 (PG-1, RGGRLCYCRRRFCVCVGR-NH2) in a fully hydrated dilauroylphosphatidylcholine (DLPC) lipid bilayer have been implemented. The goal of the reported work is to investigate the structure of the peptide in a membrane environment (previously solved only in solution [R.L. Fahrner, T. Dieckmann, S.S.L. Harwig, R.I. Lehrer, D. Eisenberg, J. Feigon, Solution structure of protegrin-1, a broad-spectrum antimicrobial peptide from porcine leukocytes. Chemistry and Biology, 3 (1996) 543-550]), and to delineate specific peptide-membrane interactions which are responsible for the peptide's membrane binding properties. A novel, previously unknown, “kick” shaped conformation of the peptide was detected, where a bend at the C-terminal β-strand of the peptide caused the peptide backbone at residues 16-18 to extend perpendicular to the β-hairpin plane. This bend was driven by a highly persistent hydrogen-bond between the polar peptide side-chain of TYR7 and the unshielded backbone carbonyl oxygen atom of GLY17. The H-bond formation relieves the unfavorable free energy of insertion of polar groups into the hydrophobic membrane core. PG-1 was anchored to the membrane by strong electrostatic binding of the protonated N-terminus of the peptide to the lipid head group phosphate anions. The orientation of the peptide in the membrane, and its influence on bilayer structural and dynamic properties are in excellent agreement with solid state NMR measurements [S. Yamaguchi, T. Hong, A. Waring, R.I. Lehrer, M. Hong, Solid-State NMR Investigations of Peptide-Lipid Interaction and Orientation of a b-Sheet Antimicrobial Peptide, Protegrin, Biochemistry, 41 (2002) 9852-9862]. Importantly, two simulations which started from different initial orientations of the peptide converged to the same final equilibrium orientation of the peptide relative to the bilayer. The kick-shaped conformation was observed only in one of the two simulations. 相似文献
6.
Summary The 15N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional 1H–15N NMR spectroscopy. Measurements of 15N longitudinal (RN(Nz)) and transverse (RN(Nx,y)) relaxation rates and the {1H} 15N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the 1H–15N lingitudinal two-spin order, RNH(2H
infZ
supN
NZ), two-spin antiphase coherence, RNH(2H
infN
supZ
Nx,y), and the proton longitudinal relaxation rate, RH(H
infN
supZ
), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide 15N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed. 相似文献
7.
8.
Iwadate M Asakura T Dubovskii PV Yamada H Akasaka K Williamson MP 《Journal of biomolecular NMR》2001,19(2):115-124
A novel method is described, which uses changes in NMR chemical shifts to characterise the structural change in a protein with pressure. Melittin in methanol is a small -helical protein, and its chemical shifts change linearly and reversibly with pressure between 1 and 2000 bar. An improved relationship between structure and HN shift has been calculated, and used to drive a molecular dynamics-based calculation of the change in structure. With pressure, the helix is compressed, with the H—O distance of the NH—O=C hydrogen bonds decreased by 0.021 ± 0.039 Å, leading to an overall compression along the entire helix of about 0.4 Å, corresponding to a static compressibility of 6 ×10–6 bar–1. The backbone dihedral angles and are altered by no more than ± 3° for most residues with a negative correlation coefficient of –0.85 between i and i–1, indicating that the local conformation alters to maintain hydrogen bonds in good geometries. The method is shown to be capable of calculating structural change with high precision, and the results agree with structural changes determined using other methodologies. 相似文献
9.
Holger A. Scheidt Robert M. Badeau Daniel Huster 《Chemistry and physics of lipids》2010,163(4-5):356-361
17β-Estradiol (E2) is a potent estrogen, which modulates many important cellular functions by binding to specific estrogen receptors located in the cell nucleus and also on the plasma membrane. We have studied the membrane interaction of E2 using a combination of solid-state NMR methods. 2H NMR results indicate that E2 does not cause a condensation effect of the surrounding phospholipids, which is contrary to the effects of cholesterol, and only very modest E2 induced alterations of the membrane structure were detected. 1H magic-angle spinning NMR showed well resolved signals from E2 as well as of POPC in the membrane-lipid layer. Two-dimensional NOESY spectra revealed intense cross-peaks between E2 and the membrane lipids indicating that E2 is stably inserted into the membrane. The determination of intermolecular cross-relaxation rates revealed that E2 is broadly distributed in the membrane with a maximum of the E2 distribution function in the upper chain region of the membrane. We conclude that E2 is highly dynamic in lipid membranes and may undergo rotations as it exhibits two polar hydroxyl groups on either side of the molecule. 相似文献
10.
-Conotoxin EI is an 18-residue peptide (RDOCCYHPTCNMSNPQIC; 4–10, 5–18) isolated from the venom of Conus ermineus, the only fish-hunting cone snail of the Atlantic Ocean. This peptide targets specifically the nicotinic acetylcholine receptor (nAChR) found in mammalian skeletal muscle and the electric organ Torpedo, showing a novel selectivity profile when compared to other -conotoxins. The 3D structure of EI has been determined by 2D-NMR methods in combination with dynamical simulated annealing protocols. A total of 133 NOE-derived distances were used to produce 13 structures with minimum energy that complied with the NOE restraints. The structure of EI is characterized by a helical loop between Thr9 and Met12 that is stabilized by the Cys4-Cys10 disulfide bond and turns involving Cys4-Cys5 and Asn14-Pro15. Other regions of the peptide appear to be flexible. The overall fold of EI is similar to that of other 4/7-conotoxins (PnIA/B, MII, EpI). However, unlike these other 4/7-conotoxins, EI targets the muscular type nAChR. The differences in selectivity can be attributed to differences in the surface charge distribution among these 4/7-conotoxins. The implications for binding of EI to the muscular nAChR are discussed with respect to the current NMR structure of EI. 相似文献
11.
Antoine Gautier 《BBA》2014
The biochemical processes of living cells involve a numerous series of reactions that work with exceptional specificity and efficiency. The tight control of this intricate reaction network stems from the architecture of the proteins that drive the chemical reactions and mediate protein–protein interactions. Indeed, the structure of these proteins will determine both their function and interaction partners. A detailed understanding of the proximity and orientation of pivotal functional groups can reveal the molecular mechanistic basis for the activity of a protein. Together with X-ray crystallography and electron microscopy, NMR spectroscopy plays an important role in solving three-dimensional structures of proteins at atomic resolution. In the challenging field of membrane proteins, retinal-binding proteins are often employed as model systems and prototypes to develop biophysical techniques for the study of structural and functional mechanistic aspects. The recent determination of two 3D structures of seven-helical trans-membrane retinal proteins by solution-state NMR spectroscopy highlights the potential of solution NMR techniques in contributing to our understanding of membrane proteins. This review summarizes the multiple strategies available for expression of isotopically labeled membrane proteins. Different environments for mimicking lipid bilayers will be presented, along with the most important NMR methods and labeling schemes used to generate high-quality NMR spectra. The article concludes with an overview of types of conformational restraints used for generation of high-resolution structures of membrane proteins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks. 相似文献
12.
Mallikarjunaiah KJ Leftin A Kinnun JJ Justice MJ Rogozea AL Petrache HI Brown MF 《Biophysical journal》2011,100(1):98-107
Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state 2H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our 2H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state 2H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state 2H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins. 相似文献
13.
Lv G Kumar A Giller K Orcellet ML Riedel D Fernández CO Becker S Lange A 《Journal of molecular biology》2012,420(1-2):99-111
Fibrillar α-synuclein (AS) is the major component of Lewy bodies, the pathological hallmark of Parkinson's disease. Mouse AS (mAS) aggregates much faster than human AS (hAS), although mAS differs from hAS at only seven positions in its primary sequence. Currently, little is known about the site-specific structural differences between mAS and hAS fibrils. Here, we applied state-of-the-art solid-state nuclear magnetic resonance (ssNMR) methods to structurally characterize mAS fibrils. The assignment strategy employed a set of high-resolution 2D and 3D ssNMR spectra recorded on uniformly [(13)C, (15)N], [1-(13)C]glucose, and [2-(13)C]glucose labeled mAS fibrils. An almost complete resonance assignment (96% of backbone amide (15)N and 93% of all (13)C nuclei) was obtained for residues from Gly41 to Val95, which form the core of mAS fibrils. Six β-strands were identified to be within the fibril core of mAS based on a secondary chemical shift and NHHC analysis. Intermolecular (13)C:(15)N labeled restraints obtained from mixed 1:1 (13)C/(15)N-labeled mAS fibrils reveal a parallel, in-register supramolecular β-sheet arrangement. The results were compared in detail to recent structural studies on hAS fibrils and indicate the presence of a structurally conserved motif comprising residues Glu61-Lys80. 相似文献
14.
《Inorganica chimica acta》1986,122(1):45-53
The structure and bonding properties of a number of closely related tetraphenyltin- and triphenyltin chloride compounds have been studied by the 119Sn Mössbauer effect and multinuclear NMR spectroscopy. The comparison of liquid and solid state 13C and 119Sn NMR spectra and of glassy solution matrix and neat solid state Mössbauer spectra provides information about the extent of intermolecular association effects in these compounds. The results indicate that all materials with the exception of (p-CF3Ph)3SnCl are adequately described as monomeric solids with tetrahedral geometry around the metal atom. For the latter compound spectroscopic evidence for the presence of a five-coordinated tin species is presented. 相似文献
15.
16.
Bondarenko V Mowrey D Tillman T Cui T Liu LT Xu Y Tang P 《Biochimica et biophysica acta》2012,1818(5):1261-1268
The α4β2 nicotinic acetylcholine receptor (nAChR) is the predominant heteromeric subtype of nAChRs in the brain, which has been implicated in numerous neurological conditions. The structural information specifically for the α4β2 and other neuronal nAChRs is presently limited. In this study, we determined structures of the transmembrane (TM) domains of the α4 and β2 subunits in lauryldimethylamine-oxide (LDAO) micelles using solution NMR spectroscopy. NMR experiments and size exclusion chromatography-multi-angle light scattering (SEC-MALS) analysis demonstrated that the TM domains of α4 and β2 interacted with each other and spontaneously formed pentameric assemblies in the LDAO micelles. The Na(+) flux assay revealed that α4β2 formed Na(+) permeable channels in lipid vesicles. Efflux of Na(+) through the α4β2 channels reduced intra-vesicle Sodium Green? fluorescence in a time-dependent manner that was not observed in vesicles without incorporating α4β2. The study provides structural insight into the TM domains of the α4β2 nAChR. It offers a valuable structural framework for rationalizing extensive biochemical data collected previously on the α4β2 nAChR and for designing new therapeutic modulators. 相似文献
17.
Chevelkov V Faelber K Diehl A Heinemann U Oschkinat H Reif B 《Journal of biomolecular NMR》2005,31(4):295-310
Water molecules are a major determinant of protein stability and are important for understanding protein–protein interactions. We present two experiments which allow to measure first the effective T2 decay rate of individual amide proton, and second the magnetization build-up rates for a selective transfer from H2O to HN using spin diffusion as a mixing element. The experiments are demonstrated for a uniformly 2H, 15N labeled sample of a microcrystalline SH3 domain in which exchangeable deuterons were back-substituted with protons. In order to evaluate the NMR experimental data, as X-ray structure of the protein was determined using the same crystallization protocol as for the solid-state NMR sample. The NMR experimental data are correlated with the dipolar couplings calculated from H2O–HN distances which were extracted from the X-ray structure of the protein. We find that the HN T2 decay rates and H2O–HN build-up rates are sensitive to distance and dynamics of the detected water molecules with respect to the protein. We show that qualitative information about localization and dynamics of internal water molecules can be obtained in the solid-state by interpretation of the spin dynamics of a reporter amide proton. 相似文献
18.
Jee-Young Lee Sung-Ah Lee Jin-Kyoung Kim Chi-Bom Chae Yangmee Kim 《Molecules and cells》2009,27(6):651-656
The formation of β-amyloid peptide (Aβ) is initiated from cleavage of amyloid precursor protein (APP) by a family of protease,
α-, β-, and γ-secretase. Sub W, a substrate peptide, consists of 10 amino acids, which are adjacent to the β-cleavage site
of wild-type APP, and Sub M is Swedish mutant with double mutations on the left side of the β-cleavage site of APP. Sub W
is a normal product of the metabolism of APP in the secretary pathway. Sub M is known to increase the efficiency of β-secretase
activity, resulting in a more specific binding model compared to Sub W. Three-dimensional structures of Sub W and Sub M were
studied by CD and NMR spectroscopy in water solution. On the basis of these structures, interaction models of β-secretase
and substrate peptides were determined by molecular dynamics simulation. Four hydrogen bonds and one water-mediated interaction
were formed in the docking models. In particular, the hydrogen bonding network of Sub M-BACE formed spread over the broad
region of the active site of β-secretase (P5-P3′), and the side chain of P2-Asn formed a hydrogen bond specifically with the
side chain of Arg235. These are more favorable to the cleavage of Sub M by β-secretase than Sub W. The two substrate peptides
showed different tendency to bind to β-secretase and this information may useful for drug development to treat and prevent
Alzheimer’s disease. 相似文献
19.
Alessandro Grottesi Marco Sette Anna Teresa Palamara Giuseppe Rotilio Enrico Garaci Maurizio Paci 《Peptides》1998,19(10):1731-1738
The 28-residue peptide thymosin α1 was studied by circular dichroism and two-dimensional NMR. Circular dichroism indicates that thymosin α1 in water solution does not assume a preferred conformation, while in the presence of small unilamellar vesicles of dimiristoylphosphatidylcholine and dimiristoylphosphatidic acid (10:1) and in sodium dodecyl sulphate, it assumes a partly structured conformation. Presence of zinc ions produces similar effects. In a more hydrophobic environment like a solution of a mixed solvent water-2,2,2 trifluoroethanol, it adopts a structured conformation. NMR spectra indicated that in this mixture as solvent, thymosin α1 has a structure characterized by two regions. A β-turn is present between residue 5 and residue 8, while the region between residues 17 and 24 shows an α helix conformation. These changes of conformation in different environments may be considered structural requirements in the steps of its interaction with the lymphocyte membrane. In fact, these conformational changes may correspond to the first event of the mechanism of lymphocyte activation in the immune response modulation by thymosin α1. 相似文献
20.
Summary Fluorine NMR lineshape, relaxation and Overhauser effect data collected at 282 and 470 MHz have been used to obtain information about the nature of complexes formed betweenN-trifluoroacetyl-4-fluorophenylalanine and the enzyme chymotrypsin. Systems involving both enantiomers have been examined as well as derivatives of these in which the aromatic ring hydrogens have been replaced by deuterium. The enzyme-induced fluorine chemical shift effects and the dynamics of molecular motions of the fluorophenyl ring at the respective binding sites appear to be similar in both complexes and, where comparable, the results are in agreement with data obtained at lower frequencies that have been reported by other workers. The dynamics of the fluoroaromatic ring in these complexes are significantly different from those observed in a closely related acylated enzyme. 相似文献