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1.
The orientations of the retinyl and heme chromophores of bacteriorhodopsin and cytochrome b-561 of the brown membrane of Halobacterium halobium have been determined by linear dichroic spectroscopy of oriented brown membrane films. Both chromophores exhibit cylindrical symmetry with respect to the membrane normal. However, the retinyl transition dipole moment is polarized at an angle of 20 to 24 ° with respect to the plane of the membrane while the plane of the heme is oriented nearly perpendicular to the membrane plane. Therefore, the orientation of retinal bound to bacterio-opsin in the brown membrane is approximately the same as in the purple membrane. This is supportive of our previous conclusions that the fine structures of the bacteriorhodopsins of these membranes are very similar in spite of differences in the composition and structure of the two membranes. The orientation of the heme plane of the membrane-bound cytochrome b-561 is very similar to orientations of several membrane-bound heme proteins that are involved in electron transfer processes and may be suggestive of its function in the brown membrane. Analysis of the linear dichroic spectrum over the entire bacteriorhodopsin band using an exciton formalism is in accord with the energy separation of the in-plane and out-of-plane excitonic transitions being less than 5 nm. Since a similar energy separation was reported for the purple membrane, the relative positions of the retinals must be approximately the same in both membranes. A similar analysis of the Soret region, based on the existence of two degenerate mutually perpendicular porphyrin transitions, indicates that the energy separation should be from 5 to 20 nm. However, the smaller value is unlikely for it would imply very large circular dichroic bands not yet encountered in any heme proteins.  相似文献   

2.
Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.  相似文献   

3.
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   

4.
Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a β-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.  相似文献   

5.
Conformational analysis of N-phenyl-1-naphthylamine and 1-anilinonaphthalene-8-sulfonate (ANS) was carried out using the empirical method. Properties such as conformational energies and dipole moments were considered. Furthermore, the effect of solvent medium was examined through the effective dielectic constant. The N-phenyl-1-naphthylamine molecule showed two energy minima which were independent of dielectic constant. The ANS molecule also showed two energy minima but the minima changed positions when the dielectic constant increased from 1.0 (vacuum) to 80.0 (highly polar medium). Hydrogen bonding appeared to play an important role in stabilizing these conformations. The minimum energy conformations may have relevance to the binding of ANS to lipid bilayers and bimembranes. The dipole moment, in contrast to the energy minimum, was found to depend on orientation of the sulfonate group rather than of the benzene ring with respect to the naphthalene ring. Thus binding and fluorescence enhancement of ANS may be attributed to the orientation of the sulfonate group, which to a large extent may determine the magnitude of the dipole moment and the degree of electrostatic interactions between the probe and binding domains. Various dimensions like intra-atomic distances, volume and area of the ANS molecule were calculated.  相似文献   

6.
Chicken liver bile acid-binding protein (L-BABP) is a member of the fatty acid-binding proteins super family. The common fold is a beta-barrel of ten strands capped with a short helix-loop-helix motif called portal region, which is involved in the uptake and release of non-polar ligands. Using multiple-run molecular dynamics simulations we studied the interactions of L-BABP with lipid membranes of anionic and zwitterionic phospholipids. The simulations were in agreement with our experimental observations regarding the electrostatic nature of the binding and the conformational changes of the protein in the membrane. We observed that L-BABP migrated from the initial position in the aqueous bulk phase to the interface of anionic lipid membranes and established contacts with the head groups of phospholipids through the side of the barrel that is opposite to the portal region. The conformational changes in the protein occurred simultaneously with the binding to the membrane. Remarkably, these conformational changes were observed in the portal region which is opposite to the zone where the protein binds directly to the lipids. The protein was oriented with its macrodipole aligned in the configuration of lowest energy within the electric field of the anionic membrane, which indicates the importance of the electrostatic interactions to determine the preferred orientation of the protein. We also identified this electric field as the driving force for the conformational change. For all the members of the fatty acid-binding protein family, the interactions with lipid membranes is a relevant process closely related to the uptake, release and transfer of the ligand. The observations presented here suggest that the ligand transfer might not necessarily occur through the domain that directly interacts with the lipid membrane. The interactions with the membrane electric field that determine orientation and conformational changes described here can also be relevant for other peripheral proteins.  相似文献   

7.
Recent studies have shown that the small GTPase KRAS adopts multiple orientations with respect to the plane of anionic model membranes, whereby either the three C-terminal helices or the three N-terminal β-strands of the catalytic domain face the membrane. This has functional implications because, in the latter, the membrane occludes the effector-interacting surface. However, it remained unclear how membrane reorientation occurs and, critically, whether it occurs in the cell in which KRAS operates as a molecular switch in signaling pathways. Herein, using data from a 20 μs-long atomistic molecular dynamics simulation of the oncogenic G12V-KRAS mutant in a phosphatidylcholine/phosphatidylserine bilayer, we first show that internal conformational fluctuations of flexible regions in KRAS result in three distinct membrane orientations. We then show, using single-molecule fluorescence resonance energy transfer measurements in native lipid nanodiscs derived from baby hamster kidney cells, that G12V-KRAS samples three conformational states that correspond to the predicted orientations. The combined results suggest that relatively small energy barriers separate orientation states and that signaling-competent conformations dominate the overall population.  相似文献   

8.
Rotation of fluorescent probes localized within lipid bilayer membranes   总被引:1,自引:0,他引:1  
Measurements of the steady state polarization of fluorescence from perylene and 9-vinylanthracene embedded in bilayer membranes were performed as a function of temperature. Similar measurements were made when these probes were dissolved in hydrocarbons as model solvents. The effects of cholesterol and n-alkyl alcohol additions to bilayers and head group variation were also examined. Results were expressed in terms of the average rotation rates of the probes.At 25°C, the calculated rotation rate for perylene in egg phosphatidylcholine vesicles was 275 × 106 sec?1 as compared to 2400 × 106 sec?1 for perylene in n-hexadecane. However, the activation energies for probe rotation in both environments was about 7 kcal/mole suggesting similar rotational diffusion mechanisms. Membrane microviscosity evaluations were performed according to a recently published scheme and an assessment of this method of viscosity estimation was given. The presence of an approximately equimolar amount of cholesterol impeded probe rotation (90 × 106 sec?1 at 25°C) and reduced the activation energy (4.9 kcal/mole) for probe rotation. In contrast, addition of n-alkyl alcohols to the vesicle suspension acted to increase probe rotation rates, an indication of fluidization of the membranes. This is in accord with spin label and cation permeability data for similar membranes.It was concluded that this method of probing can adequately report changes in membrane dynamic structure when these changes occur uniformly over the membrane surface. The interpretation is less clear when structural changes occur only in patches or domains of the membrane thereby producing a non-uniform surface distribution of probes.  相似文献   

9.
The conformational space of a hydrophobic peptide fragment of glycophorin A in a lipid membrane was studied with the Monte Carlo method using the solvation model described in the first communication of this series. The simulation was performed for various starting orientations of the peptide relative the membrane bilayer: outside, inside, partially immersed, and transbilayer. We showed that the membrane substantially stabilizes the α-helical conformation of the central hydrophobic part of the glycophorin A molecule, which for the most part is immersed in the apolar core of the bilayer. For various conformational states, energy values were calculated and the orientations of the peptide relative to the membrane were characterized. Depending on the thickness of the bilayer, either an entirely α-helical conformation in transbilayer orientation or a conformation with a kink in the central part of the helix with theN- andC-termini exposed on one side of the membrane corresponds to the minimal-energy structure. The transmembrane orientation of glycophorin A is energetically advantageous when the membrane thickness is close to the length of its hydrophobic helical portion, which is consistent with the effect ofhydrophobic match observed experimentally. The prospects for further refinement of the model are discussed. For communication I, see [1].  相似文献   

10.
Eliane Nabedryk  Jacques Breton 《BBA》1981,635(3):515-524
In order to estimate the degree of orientation of the α-helices of intrinsic proteins in photosynthetic membranes, polarized infrared spectroscopy has been used to measure the dichroism of the amide I and amide II absorption bands of air-dried oriented samples of purple membranes, chloroplasts and chromatophores from Rhodopseudomonas sphaeroides. Using purple membrane, in which the orientation of the α-helices is precisely known (Henderson, R. (1977) Annu. Rev. Biophys. Bioeng. 6, 87–109), as a standard to calibrate our measurements and estimating the mosaic spread (extent of orientation) of the membranes from linear dichroism measurements performed in the visible spectral range, it is concluded that in photosynthetic membranes, the α-helices of intrinsic proteins are tilted at less than 40° with respect to the normal to the plane of the membrane.  相似文献   

11.
The conformational space of a hydrophobic peptide fragment of glycophorin A in a lipid membrane was studied with the Monte Carlo method using the solvation model described in the first communication of this series. The simulation was performed for various starting orientations of the peptide relative to the membrane bilayer: outside, inside, partially immersed, and transbilayer. We showed that the membrane substantially stabilizes the alpha-helical conformation of the central hydrophobic part of the glycophorin A molecule, which for the most part is immersed in the apolar core of the bilayer. For various conformational states, energy values were calculated and the orientations of the peptide relative to the membrane were characterized. Depending on the thickness of the bilayer, either an entirely alpha-helical conformation in transbilayer orientation or a conformation with a kink in the central part of the helix with the N- and C-termini exposed on one side of the membrane corresponds to the minimal-energy structure. The transmembrane orientation of glycophorin A is energetically advantageous when the membrane thickness is close to the length of its hydrophobic helical portion, which is consistent with the effect of "hydrophobic match" observed experimentally. The prospects for further refinement of the model are discussed.  相似文献   

12.
Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short fange interaction we developed a spectroscopic method to characterize the molecular arrangement within the lipid membrane. Applied to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase.This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.  相似文献   

13.
Spin probes differing in the position of their paramagnetic centre are used to quench the fluorescence of pyrene derivatives and chlorophylls incorporated into dimyristoyl phosphatidylcholine membranes. Pyrene butyric acid and pyrene decanoic acid with known orientation relative to the membrane surface are investigated. The quenching efficiency of fatty acid spin probes is dependent on the position of the nitroxide radical group in the fatty acid chain. Using this short range interaction we developed a spectroscopic method to chlorophyll-containing vesicles, we were able to characterize the orientation of the porphyrin ring within the membrane. Moreover, the chlorophyll fluorescence is also quenched by a water-soluble spin label. Therefore the porphyrin ring appears to be orientated in the polar head group region of the lipid layer, but not to be protruding out into the water phase. This conclusion is confirmed by the use of pyrene derivatives. Fluorescence quenching by a water-soluble spin label within the lipid matrix is observed even in the rigid state of the membrane. Fluorescence lifetime measurements suggest the existence of two different quenching mechanisms: (1) a static quenching occurring below the lipid phase transition temperature, and (2) an additional dynamic quenching taking place in the fluid state of the lipid bilayer.  相似文献   

14.
We introduce here a novel Monte Carlo simulation method for studying the interactions of hydrophobic peptides with lipid membranes. Each of the peptide's amino acids is represented as two interaction sites: one corresponding to the backbone alpha-carbon and the other to the side chain, with the membrane represented as a hydrophobic profile. Peptide conformations and locations in the membrane and changes in the membrane width are sampled using the Metropolis criterion, taking into account the underlying energetics. Using this method we investigate the interactions between the hydrophobic peptide M2delta and a model membrane. The simulations show that starting from an extended conformation in the aqueous phase, the peptide first adsorbs onto the membrane surface, while acquiring an ordered helical structure. This is followed by formation of a helical-hairpin and insertion into the membrane. The observed path is in agreement with contemporary understanding of peptide insertion into biological membranes. Two stable orientations of membrane-associated M2delta were obtained: transmembrane (TM) and surface, and the value of the water-to-membrane transfer free energy of each of them is in agreement with calculations and measurements on similar cases. M2delta is most stable in the TM orientation, where it assumes a helical conformation with a tilt of 14 degrees between the helix principal axis and the membrane normal. The peptide conformation agrees well with the experimental data; average root-mean-square deviations of 2.1 A compared to nuclear magnetic resonance structures obtained in detergent micelles and supported lipid bilayers. The average orientation of the peptide in the membrane in the most stable configurations reported here, and in particular the value of the tilt angle, are in excellent agreement with the ones calculated using the continuum-solvent model and the ones observed in the nuclear magnetic resonance studies. This suggests that the method may be used to predict the three-dimensional structure of TM peptides.  相似文献   

15.
Large (0.5–1.0 μm) cytoskeleton-free vesicles were obtained, by ‘budding’, from fresh human and rabbit erythrocytes incubated at 45°C and titrated with EDTA and CaCl2. This process occurs without hemolysis. The isolated vesicles maintain their cytoplasmic integrity and normal membrane orientation, and are resistant to hemolysis over the pH range 5.0–11.0 and temperature range 4–50°C. The only membrane proteins detected in vesicles from human erythrocytes were band 3 region polypeptides and bands PAS-1, PAS-2 and PAS-3. Vesicles obtained from rabbit erythrocytes were similarly simple. Because of their size and stability these vesicles are amenable to both kinetic and quantitative analysis using the same experimental techniques employed in studies of synthetic lipid membranes. The results obtained in this study indicate that these vesicles are essentially markedly simplified biological cells, and thus may be useful as a biologically relevant model membrane system for examining the molecular interactions which occur within, across and between cell membranes.  相似文献   

16.
Biological membranes are elastic media in which the presence of a transmembrane protein leads to local bilayer deformation. The energetics of deformation allow two membrane proteins in close proximity to influence each other's equilibrium conformation via their local deformations, and spatially organize the proteins based on their geometry. We use the mechanosensitive channel of large conductance (MscL) as a case study to examine the implications of bilayer-mediated elastic interactions on protein conformational statistics and clustering. The deformations around MscL cost energy on the order of 10 kBT and extend ~3 nm from the protein edge, as such elastic forces induce cooperative gating, and we propose experiments to measure these effects. Additionally, since elastic interactions are coupled to protein conformation, we find that conformational changes can severely alter the average separation between two proteins. This has important implications for how conformational changes organize membrane proteins into functional groups within membranes.  相似文献   

17.
The semiempirical CNDO/2 SCF MO method using the tight-binding approximation for polymers has been applied to poly(β-hydroxy-l-proline), β-PHP, to compare the electronic structure of β-PHP with that of poly(γ-hydroxy-l-proline), γ-PHP, which we have described in a previous publication. The results obtained show the preferred orientation of the OH group at the β-position of the pyrrolidine ring. The different situation between β-PHP and γ-PHP is briefly discussed. Analysis of the calculated results shows that the energy difference between the two species is not sufficient to deny the existence of either form. This agrees well with the experimental results. The conformational stability between the trans and cis forms of the H:C:O:H group is explained by using the calculated results in connection with the previous experimental and theoretical treatments. From the analysis of the total energy, the dominant stabilizing factors are discussed.  相似文献   

18.
Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τc) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τc at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (Ea) for Arrhenius plots of τc above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τc plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.  相似文献   

19.
The B800–850 antenna complex of Rhodopseudomonas sphaeroides was studied by comparing the spectral properties of several different types of complexes, isolated from chromatophores by means of the detergents lithium dodecyl sulfate (LDS) or lauryl dimethylamine N-oxide (LDAO). Fluorescence polarization spectra of the BChl 800 emission at 4 K indicated that rapid energy transfer between at least two BChl 800 molecules occurs with a rate constant of energy transfer kET > 3 · 1012 s?1. The maximal dipole-dipole distance between the two BChl 800 molecules was calculated to be 18–19 Å. The porphyrin rings of the BChl 800 molecules are oriented parallel to each other, while their Qy transition moments are mutually perpendicular. The energy-transfer efficiency from carotenoid to bacteriochlorophyll measured in different complexes showed that two functionally different carotenoids are present associated with, respectively, BChl 800 and BChl 850. Fluorescence polarization and linear dichroism spectra revealed that these carotenoids have different absorption spectra and a different orientation with respect to the membrane. The carotenoid associated with BChl 800 absorbs some nanometers more to the red and its orientation is approximately parallel to the membrane, while the carotenoid associated with BChl 850 is oriented more or less perpendicular to the membrane. The fluorescence polarization of BChl 850 was the same for the different complexes. This indicates that the observed polarization of the fluorescence is determined by the smallest complex obtained which contains 8–10 BChl 850 molecules. The B800–850 complex isolated with LDAO thus must consist of a highly ordered array of smaller structures. On basis of these results a minimal model is proposed for the basic unit consisting of four BChl 850 and two BChl 800 and three carotenoid molecules.  相似文献   

20.
Raman spectroscopy and X-ray diffraction are used to investigate the influence of surface charges on the structure of ionizable lipid membranes of dimyristoylmethylphosphatidic acid. The membrane surface charge density is regulated by varying the pH of the aqueous phase. Changes of the conformational order of the lipid chains are determined from the intensity of the CC stretch chain vibrations around 1100 cm?1 in a lipid Raman spectrum. In going from an electrical neutral to a negatively charged membrane, the conformational order is reduced by 5% in the ordered and by 9% in the fluid membrane phase, corresponding to 0.6 and 0.8 CC bonds, respectively, which change from a trans to a gauche conformation. The electrostatically induced conformational change is mainly concentrated at the lipid chain ends as indicated by the spectral variations of the 890 cm?1 CH3 rocking band of the chain termini. The X-ray diffraction experiments show that increasing the surface charge density in the ordered membrane phase leads to a lateral expansion of the packing of the lipid polar groups, whereas the packing of the lipid chains in a plane perpendicular to the chain axes remains constant, indicating an increase of the tilt of the lipid chains from δ = 10° (pH 3) to δ = 27° (pH 9).  相似文献   

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