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1.
The helix tilt and rotational orientation of the transmembrane segment of M2, a 97-residue protein from the Influenza A virus that forms H(+)-selective ion channels, have been determined by attenuated total reflection site-specific infrared dichroism using a novel labeling approach. Triple C-deuteration of the methyl group of alanine in the transmembrane domain of M2 was used, as such modification shifts the asymmetric and symmetric stretching vibrations of the methyl group to a transparent region of the infrared spectrum. Structural information can then be obtained from the dichroic ratios corresponding to these two vibrations. Two consecutive alanine residues were labeled to enhance signal intensity. The results obtained herein are entirely consistent with previous site-specific infrared dichroism and solid-state nuclear magnetic resonance experiments, validating C-deuterated alanine as an infrared structural probe that can be used in membrane proteins. This new label adds to the previously reported (13)C [double bond] (18)O and C-deuterated glycine as a tool to analyze the structure of simple transmembrane segments and will also increase the feasibility of the study of polytopic membrane proteins with site-specific infrared dichroism. 相似文献
2.
Polarized UV light irradiation of flow-oriented fd bacteriophage indicates that the degree of damage (inactivation) depends on the relative orientation of the light polarization vector and the plane of the DNA bases. The technique of anisotropic UV inactivation was evaluated, and further information on the orientation in this virus was gained. The fd bacteriophage were aligned and irradiated with plane-polarized monochromatic UV light either parallel or perpendicular to the virus axis. Variation of the inactivation dichroic ratio with wavelength implicated virus inactivation by light absorbed in both the DNA and protein. Analysis of the wavelength variation of inactivation dichroic ratios gave molecular dichroic ratios of 0.76 and 1.48 for the DNA and protein components, respectively. On the basis of these anisotropic inactivation studies, the average angle of DNA base tilt in fd was calculated to be 29-32°, a value in agreement with the absorption dichroism studies of Bendet and Mayfield. 相似文献
3.
The 115-residue protein CM2 from Influenza C virus has been recently characterized as a tetrameric integral membrane glycoprotein. Infrared spectroscopy and site-directed infrared dichroism were utilized here to determine its transmembrane structure. The transmembrane domain of CM2 is alpha-helical, and the helices are tilted by beta = (14.6 +/- 3.0) degrees from the membrane normal. The rotational pitch angle about the helix axis omega for the 1-(13)C-labeled residues Gly(59) and Leu(66) is omega = (218 +/- 17) degrees, where omega is defined as zero for a residue pointing in the direction of the helix tilt. A detailed structure was obtained from a global molecular dynamics search utilizing the orientational data as an energy refinement term. The structure consists of a left-handed coiled-coil with a helix crossing angle of Omega = 16 degrees. The putative transmembrane pore is occluded by the residue Met(65). In addition hydrogen/deuterium exchange experiments show that the core is not accessible to water. 相似文献
4.
Experimental results using multiple site-specific infrared dichroism have shown that, when reconstituted into lipid bilayers, the orientation of the transmembrane domain of CD3-zeta is not compatible with a dimeric right-handed model reported previously. This model, obtained using a computational approach that uses evolutionary data, is in agreement with mutagenesis data and homology modelling. This suggested that, in our experimental conditions, the oligomeric state of CD3-zeta may not be dimeric. We have explored this possibility by performing global searching molecular dynamics simulations assuming different homo-oligomeric sizes (from 2 to 6). In these simulations, the helix tilt was restrained to the average helix tilt obtained experimentally, 12 degrees. Only a left-handed tetrameric model was compatible with the experimentally observed tilt and rotational orientation of the helix, and was also the lowest-energy model amongst the candidate structures obtained. Furthermore, simulations performed using close homologues demonstrate that this model is compatible with evolutionary conservation data. Finally, the pattern of residue conservation in the zeta family of proteins strongly argues in favour of the presence of a left-handed hetero-oligomer with an orientation compatible with the tetramer we present. These results show that both the known dimeric and the so far undetected tetrameric form may be of functional importance in the cell. 相似文献
5.
The 97-residue M2 protein from Influenza A virus forms H+-selective ion channels which can be attributed solely to the homo-tetrameric alpha-helical transmembrane domain. Site-directed infrared dichroism spectra were obtained for the transmembrane domain of M2, reconstituted in lipid vesicles. Data analysis yielded the helix tilt angle beta=31.6(+/-6.2) degrees and the rotational pitch angle about the helix axis for residue Ala29 omegaAla29=-59.8(+/-9.9) degrees, whereby omega is defined as zero for a residue located in the direction of the helix tilt. A structure was obtained from an exhaustive molecular dynamics global search protocol in which the orientational data are utilised directly as an unbiased refinement energy term. Orientational refinement not only allowed selection of a unique structure but could also be shown to increase the convergence towards that structure during the molecular dynamics procedure. Encouragingly, the structure obtained is highly consistent with all available mutagenesis and conductivity data and offers a direct chemical insight that relates the altered functionality of the channel to its structure. 相似文献
6.
vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching. 总被引:1,自引:0,他引:1
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The recently developed method of site-directed Fourier transform infrared dichroism for obtaining orientational constraints of oriented polymers is applied here to the transmembrane domain of the vpu protein from the human immunodeficiency virus type 1 (HIV-1). The infrared spectra of the 31-residue-long vpu peptide reconstituted in lipid vesicles reveal a predominantly alpha-helical structure. The infrared dichroism data of the (13)C-labeled peptide yielded a helix tilt beta = (6.5 +/- 1.7) degrees from the membrane normal. The rotational pitch angle omega, defined as zero for a residue located in the direction of the helix tilt, is omega = (283 +/- 11) degrees for the (13)C labels Val(13)/Val(20) and omega = (23 +/- 11) degrees for the (13)C labels Ala(14)/Val(21). A global molecular dynamics search protocol restraining the helix tilt to the experimental value was performed for oligomers of four, five, and six subunits. From 288 structures for each oligomer, a left-handed pentameric coiled coil was obtained, which best fits the experimental data. The structure reveals a pore occluded by Trp residues at the intracellular end of the transmembrane domain. 相似文献
7.
Infrared spectroscopy study on the conformational changes leading to pore formation of the toxin sticholysin II
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Alegre-Cebollada J Martínez del Pozo A Gavilanes JG Goormaghtigh E 《Biophysical journal》2007,93(9):3191-3201
The structure of the actinoporin sticholysin II (StnII) in the pore state was investigated by Fourier transform infrared spectroscopy in the attenuated total reflection configuration. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol unilamellar vesicles were employed. The alpha-helix content increases in approximately 30% upon lipid binding, which agrees with an extension of eight or nine residues at the N-terminal helix. Furthermore, analyses of dichroic spectra show that the extended N-terminal helix would have a 31 degrees tilt with respect to the membrane normal. The orientation of the central beta-sandwich was also estimated. In addition, it was detected that StnII alters the orientation of the lipid acyl chains. (1)H/(2)H exchange experiments sustain a mainly superficial interaction between StnII and the membrane, with no protection of the beta-sandwich. The implications of the results in the mechanism of pore formation are discussed. 相似文献
8.
Films of DNA–dye complexes were combined with films of pure DNA deposited on poly(vinyl alcohol) support and stretched. Reproducible dichroic spectra were obtained after equilibration of the stretched films at 93% relative humidity. Dye diffusion into the supporting poly(vinl alcohol) matrix was eliminated. The long axis of intercalated acriflavine is perpendicular to the DNA helix; proflavine deviates slightly and 9-aminoacridine significantly from such an intercalation geometry. The dichroism of two mutually perpendicularly polarized transitions of 9-aminoacridine enabled us to determine both the angles of tilt and twist of the plane of the dye relative to the DNA helix in the complex. 相似文献
9.
A detailed comparison with the three-dimensional protein structure provides a stringent test of the models and parameters commonly used in determining the orientation of the alpha-helices from the linear dichroism of the infrared amide bands, particularly in membranes. The order parameters of the amide vibrational transition moments are calculated for the transmembrane alpha-helices of bacteriorhodopsin by using the crystal structure determined at a resolution of 1.55 A (PDB accession number 1C3W). The dependence on the angle delta(M) that the transition moment makes with the peptide carbonyl bond is fit by the expression ((3)/(2)S(alpha) cos(2) alpha)cos(2)(delta(M) + beta) - 1/2S(alpha), where S(alpha) (0.91) is the order parameter of the alpha-helices, alpha (13 degrees ) is the angle that the peptide plane makes with the helix axis, and beta (11 degrees ) is the angle that the peptide carbonyl bond makes with the projection of the helix axis on the peptide plane. This result is fully consistent with the model of nested axial distributions commonly used in interpreting infrared linear dichroism of proteins. Comparison with experimental infrared dichroic ratios for bacteriorhodopsin yields values of Theta(A) = 33 +/- 1 degree, Theta(I) = 39.5 +/- 1 degree, and Theta(II) = 70 +/- 2 degrees for the orientation of the transition moments of the amide A, amide I, and amide II bands, respectively, relative to the helix axis. These estimates are close to those found for model alpha-helical polypeptides, indicating that side-chain heterogeneity and slight helix imperfections are unlikely to affect the reliability of infrared measurements of helix orientations. 相似文献
10.
R Buchet S Varga N W Seidler E Molnar A Martonosi 《Biochimica et biophysica acta》1991,1068(2):201-216
The mean orientations of the transition dipole moments associated with vibrational modes of the proteins and phospholipids of sarcoplasmic reticulum were determined on dry and hydrated membrane multilayers deposited on germanium or zinc selenide crystals, using polarized infrared attenuated total reflectance spectroscopy (P-IR-ATR). For preservation of the enzymatic activity of the Ca(2+)-ATPase the films were prepared from solutions containing 0.05 M KCl, 5 mM imidazole (pH 7.4), 0.5 mM MgCl2, 1-10 mM trehalose and dithiothreitol. The anisotropy was highest in dry films containing congruent to 7.5 micrograms protein/cm2, and decreased with increasing membrane thickness or hydration. The dichroic ratio of the CH2 vibrations (2923 cm-1) of extracted sarcoplasmic reticulum phospholipids on Ge plate was 1.56, compared with a dichroic ratio of 1.68 obtained on dry films of whole sarcoplasmic reticulum. The dichroic ratios of the amide I band (1650 cm-1) of the Ca(2+)-ATPase in the Ca2-E1 state and in the EGTA and vanadate stabilized E2-V state were nearly identical (1.60 vs. 1.62). The dichroism of the amide I, amide II and lipid CH2 vibrations was not affected by changes in the concentration of KCl (25-100 mM) or Ca2+ (approximately equal to 10(-8)-10(-4) M) and by the addition of vanadate (1 mM) or Pi (5 mM) in a calcium-free medium containing 0.5 mM EGTA. The dichroic ratio of the C-C (1033 cm-1) or CO stretching band (1046 cm-1) of trehalose incorporated into SR films was 1.2 on Ge plate; this corresponds to a mean angle of approximately 70 degrees between the plane of the trehalose ring and the normal of the film plane, suggesting that the trehalose molecules are surprisingly well oriented in the polar headgroup region of the phospholipids. The orientation of the trehalose was not affected by the presence of Ca(2+)-ATPase. 相似文献
11.
The outer-membrane proteins OmpA and FhuA of Escherichia coli are monomeric beta-barrels of widely differing size. Polarized attenuated total reflection infrared spectroscopy has been used to determine the orientation of the beta-barrels in phosphatidylcholine host matrices of different lipid chain lengths. The linear dichroism of the amide I band from OmpA and FhuA in hydrated membranes generally increases with increasing chain length from diC(12:0) to diC(17:0) phosphatidylcholine, in both the fluid and gel phases. Measurements of the amide I and amide II dichroism from dry samples are used to deduce the strand tilt (beta = 46 degrees for OmpA and beta = 44.5 degrees for FhuA). These values are then used to deduce the order parameters, P(2)(cos alpha), of the beta-barrels from the amide I dichroic ratios of the hydrated membranes. The orientational ordering of the beta-barrels and their assembly in the membrane are discussed in terms of hydrophobic matching with the lipid chains. 相似文献
12.
13.
The transmembrane (TM) domain of the M2 channel protein from influenza A is a homotetrameric bundle of α-helices and provides a model system for computational approaches to self-assembly of membrane proteins. Coarse-grained molecular dynamics (CG-MD) simulations have been used to explore partitioning into a membrane of M2 TM helices during bilayer self-assembly from lipids. CG-MD is also used to explore tetramerization of preinserted M2 TM helices. The M2 helix monomer adopts a membrane spanning orientation in a lipid (DPPC) bilayer. Multiple extended CG-MD simulations (5 × 5 μs) were used to study the tetramerization of inserted M2 helices. The resultant tetramers were evaluated in terms of the most populated conformations and the dynamics of their interconversion. This analysis reveals that the M2 tetramer has 2× rotationally symmetrical packing of the helices. The helices form a left-handed bundle, with a helix tilt angle of ∼16°. The M2 helix bundle generated by CG-MD was converted to an atomistic model. Simulations of this model reveal that the bundle's stability depends on the assumed protonation state of the H37 side chains. These simulations alongside comparison with recent x-ray (3BKD) and NMR (2RLF) structures of the M2 bundle suggest that the model yielded by CG-MD may correspond to a closed state of the channel. 相似文献
14.
S Kimura D Erne R Schwyzer 《International journal of peptide and protein research》1992,39(5):431-442
The enhancement of fluorescence emission from the tryptophan residue of glucagon, the quenching of that emission with acrylamide and with 5-doxyl and 16-doxyl stearic acid, circular dichroism spectra, the release of 6-carboxyfluorescein, and polarized infrared attenuated total reflection (IR-ATR) spectra were used to study the interaction of glucagon with intact lipid vesicles and flat bilayers. Dimyristoylphosphatidylcholine bound the peptide only below the main transition temperature, thus confirming earlier results of Epand et al. (1977). However, the peptide is also bound by vesicles of unsaturated lipids above their transition temperature, suggesting an influence of lipid area on the binding process. Circular dichroism showed that binding to such vesicles also increases the helix content of glucagon. The IR-ATR study and a comparison with dynorphin-A-(1-13)-tridecapeptide revealed profound differences in orientation of the two peptides. The dichroic ratios and the derived order parameters indicated an isotropic orientation of the helical segments of glucagon, but did not exclude a principal orientation of the molecules lying flat on the membrane surface. In contrast, the axis of the dynorphin helix is clearly oriented normal to the interface. The two peptides also differ in their rates of 6-carboxyfluorescein release, suggesting a deeper penetration of the primary amphiphilic helix of dynorphin A-(1-13) than of the secondary amphiphilic helix of glucagon. 相似文献
15.
Holdbrook DA Leung YM Piggot TJ Marius P Williamson PT Khalid S 《Biochemistry》2010,49(51):10796-10802
The N-terminal domain of fukutin-I has been implicated in the localization of the protein in the endoplasmic reticulum and Golgi Apparatus. It has been proposed to mediate this through its interaction with the thinner lipid bilayers found in these compartments. Here we have employed multiscale molecular dynamics simulations and circular dichroism spectroscopy to explore the structure, stability, and orientation of the short 36-residue N-terminus of fukutin-I (FK1TMD) in lipids with differing tail lengths. Our results show that FK1TMD adopts a stable helical conformation in phosphatidylcholine lipids when oriented with its principal axis perpendicular to the bilayer plane. The stability of the helix is largely insensitive to the lipid tail length, preventing hydrophobic mismatch by virtue of its mobility and ability to tilt within the lipid bilayers. This suggests that changes in FK1TMD tilt in response to bilayer properties may be implicated in the regulation of its trafficking. Coarse-grained simulations of the complex Golgi membrane suggest the N-terminal domain may induce the formation of microdomains in the surrounding membrane through its preferential interaction with 1,2-dipalmitoyl-sn-glycero-3-phosphatidylinositol 4,5-bisphosphate lipids. 相似文献
16.
Transient and linear dichroism studies on bacteriorhodopsin: determination of the orientation of the 568 nm all-trans retinal chromophore 总被引:22,自引:0,他引:22
The orientation of the 568 nm transition dipole moment of the retinal chromophore of bacteriorhodopsin has been determined in purple membranes from Halobacterium halobium and in reconstituted vesicles. The angle between the 568 nm transition dipole moment and the normal to the plane of the membrane was measured in two different ways.In the first method the angle was obtained from transient dichroism measurements on bacteriorhodopsin incorporated into large phosphatidylcholine vesicles. Following flash excitation with linearly polarized light, the anisotropy of the 568 nm ground-state depletion signal first decays but then reaches a time-independent value. This result, obtained above the lipid phase transition, is interpreted as arising from rotational motion of bacteriorhodopsin which is confined to an axis normal to the plane of the membrane. It is shown that the relative amplitude of the time-independent component depends on the orientation of the 568 nm transition dipole moment. From the data an angle of 78 ° ± 3 ° is determined.In the second method the linear dichroism was measured as a function of the angle of tilt between the oriented purple membranes and the direction of the light beam. The results were corrected for the angular distribution of the membranes within the oriented samples, which was determined from the mosaic spread of the first-order lamellar neutron diffraction peak. In substantial agreement with the results of the transient dichroism method, linear dichroism measurements on oriented samples lead to an angle of 71 ° ± 4 °.No significant wavelength dependence of the dichroic ratio across the 568 nm band was observed, implying that the exciton splitting in this band must be substantially smaller than the recently suggested value of 20 nm (Ebrey et al., 1977).The orientation of the 568 nm transition dipole moment, which coincides with the direction of the all-trans polyene chain of retinal, is not only of interest in connection with models for the proton pump, but can also be used to calculate the inter-chromophore distances in the purple membrane. 相似文献
17.
The conformational dynamic capabilities of the in situ bacteriorhodopsin (bR) can be studied by determination of the changes of the bR net helical segmental tilt angle (the angle between the polypeptide segments and the membrane normal) induced by various perturbations of the purple membrane (PM). The analysis of the far-UV oriented circular dichroism (CD) of the PM provides one means of achieving this. Previous CD studies have indicated that the tilt angle can change from approximately 10 degrees to 39 degrees depending on the perturbants used with no changes in the secondary structure of the bR. A recent study has indicated that the bleaching-induced tilt angle can be enhanced from approximately 24 degrees to 39 degrees by cross-linkage and papain-digestion perturbations which by themselves do not alter the tilt angle. To add further credence, this study has been repeated using midinfrared (IR) linear dichroic spectral analysis. In contrast to the CD method, analysis by the IR method depends on the orientation of the amide plane of the helix assumed. Excellent consistency is achieved between the two methods only when it is assumed that the structural characteristics of the alpha-helices of the bR are equally alpha I and alpha II in nature. Furthermore, the analysis of the IR data becomes essentially independent of the three amide transitions utilized. The net tilt angle of segments completely randomized relative to the incident light must be 54.736 in view of helix symmetry. A value of 54.735 degrees +/- 0.001 degree was achieved by the IR method for the ethanol-treated PM film, establishing this kind of film as an ideal random state standard and demonstrating the accuracy potential of the IR method. 相似文献
18.
《Molecular membrane biology》2013,30(7):360-369
AbstractThe role of histidine in channel-forming transmembrane (TM) helices was investigated by comparing the TM helices from Virus protein ‘u' (Vpu) and the M2 proton channel. Both proteins are members of the viroporin family of small membrane proteins that exhibit ion channel activity, and have a single TM helix that is capable of forming oligomers. The TM helices from both proteins have a conserved tryptophan towards the C-terminus. Previously, alanine 18 of Vpu was mutated to histidine in order to artificially introduce the same HXXXW motif that is central to the proton channel activity of M2. Interestingly, the mutated Vpu TM resulted in an increase in helix tilt angle of 11° in lipid bilayers compared to the wild-type Vpu TM. Here, we find the reverse, when histidine 37 of the HXXXW motif in M2 was mutated to alanine, it decreased the helix tilt by 10° from that of wild-type M2. The tilt change is independent of both the helix length and the presence of tryptophan. In addition, compared to wild-type M2, the H37A mutant displayed lowered sensitivity to proton concentration. We also found that the solvent accessibility of histidine-containing M2 is greater than without histidine. This suggests that the TM helix may increase the solvent exposure by changing its tilt angle in order to accommodate a polar/charged residue within the hydrophobic membrane region. The comparative results of M2, Vpu and their mutants demonstrated the significance of histidine in a transmembrane helix and the remarkable plasticity of the function and structure of ion channels stemming from changes at a single amino acid site. 相似文献
19.
Geoffrey Dougherty 《Journal of mathematical biology》1982,15(3):275-291
The optical linear dichroism of DNA-drug fibres and films can provide valuable information on the geometry of the binding and its extent, especially when used in conjunction with X-ray diffraction data from the same specimens. We have considered the macroscopic orientation of the helices within a fibre or film to be characterized by a Gaussian distribution of the helix axes about the fibre (or film) axis. Using this model we have obtained analytical expressions for the dichroic ratio without resorting to computer simulation techniques or numerical integration methods, and used them to interpret the results of experiments using DNA-phenanthridine fibres. As the humidity is increased, ethidium and dimidium bromide show an increased fraction of binding perpendicular to the helix axis, consistent with intercalation. Prothidium shows little preferred orientation in its binding, and the occurrence of a significant proportion of intercalation can be excluded. 相似文献
20.
Wu CW Cheng SF Huang WN Trivedi VD Veeramuthu B Assen B K Wu WG Chang DK 《Biochimica et biophysica acta》2003,1612(1):41-51
Mutations of the glycine residue at the amino terminus of HA2 have been shown to have a large effect on the fusion activity of HA2, the extent of which apparently correlates with the side chain bulkiness of the substituting amino acids. To investigate into the cause of abrogation in fusogenicity and virus-promoted fusion mechanism, we synthesized several peptides in which this glycine was substituted by serine, glutamic acid, or lysine. 1,2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dimyristoyl sn-glycero-3-phosphoglycerol (DMPG) were used as model membranes in the fluorescence, circular dichroism (CD), and FTIR measurements while sodium dodecyl sulfate was used in NMR studies. We found that, for the less active variants, affinity to membrane, degree of solvent dehydration, lipid perturbation, depth of insertion, and helicity were less. Comparison of affinity to membrane bilayer among these analogs revealed that binding of the fusion peptide is determined largely by the hydrophobic effect. Additionally, the orientation is closer to the membrane normal for the wild-type fusion peptide in the helix form while the inactive analogs inserted more parallel to the membrane surface. 相似文献