Simulation of electron spin resonance spectra of spin-labeled fatty acids covalently attached to the boundary of an intrinsic membrane protein. A chemical exchange model (original) (raw)
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Analysis of protein-lipid interactions based on model simulations of electron spin resonance spectra
The Journal of Physical Chemistry, 1984
ESR spectra from protein-containing lipid dispersions have been interpreted in the past primarily in terms of two nitroxide species related respectively to "fluid" and "immobile" phospholipid environments. In this report we consider interpretations based primarily on a single type of lipid, for the two cases of spin-probe-doped membranes and of chemically labeled membrane proteins. The doped membranes are viewed as bilayer fragments with considerable local order but with macroscopic disorder of such fragments in the dispersion. In this "one-site" model all lipids are similar. They are fluid and oriented in their local environment. This model does not require a second species immobilized by contact with the proteins. The labeled proteins are considered as very slowly reorienting macromolecular complexes such that the dynamic effects on the ESR spectrum arise mainly from faster internal processes. The importance of, and the potential inherent in, detailed spectral simulation based on well-conceived models are emphasized by illustrating the great range of spectral line shapes that these models can yield with suitable parameters. Simulations are compared with some recent experimental examples previously interpreted in terms of a two-site model. Reasonably good results are obtained for spin-probe-doped membranes when allowance is made for local ordering as well as for some distortion of the alkyl chain ends. Such effects are modeled by introducing an ordering potential X and a diffusion tilt angle 9 which describes the tilt of the nitroxide moiety relative to the rest of the alkyl chain. The effects of adding protein or of lowering the temperature are modeled by increasing the local ordering while decreasing somewhat the motional rates with some increase in alkyl chain distortions. In the case of spin-labeled membrane proteins, the model of very anisotropic rotation, in which the side chain containing the nitroxide is rotating more rapidly than the protein about an effective axis tilted relative to the NO axis, is found to account for the extra splittings with just a single-site model.
Biophysical Journal, 1994
Electron spin resonance (ESR) studies have been performed on spin-labeled model membranes aligned using the isopotential spin-dry ultracentrifugation (ISDU) method of Clark and Rothschild. This method relies on sedimentation of the membrane fragments onto a gravitational isopotential surface with simultaneous evaporation of the solvent in a vacuum ultracentrifuge to promote alignment. The degree of alignment obtainable using ISDU, as monitored by ESR measurements of molecular ordering for both lipid (16-PC) and cholestane spin labels (CSL), in dipalmitoylphosphatidylcholine (DPPC) model membranes compares favorably with that obtainable by pressure-annealing. The much gentler conditions under which membranes may be aligned by ISDU greatly extends the range of macroscopically aligned membrane samples that may be investigated by ESR. We report the first ESR study of an integral membrane protein, bacteriorhodopsin (BR) in well-aligned multilayers. We have also examined ISDU-aligned DPPC multilayers incorporating a short peptide gramicidin A' (GA), with higher water content than previously studied. 0.24 mol % BR/DPPC membranes with CSL probe show two distinct components, primarily in the gel phase, which can be attributed to bulk and boundary regions of the bilayer. The boundary regions show sharply decreased molecular ordering and spectral effects comparable to those observed from 2 mol % GAIDPPC membranes. The boundary regions for both BR and GA also exhibit increased fluidity as monitored by the rotational diffusion rates. The high water content of the GA/DPPC membranes reduces the disordering effect as evidenced by the reduced populations of the disordered components. The ESR spectra obtained slightly below the main phase transition of DPPC from both the peptideand proteincontaining membranes reveals a new component with increased ordering of the lipids associated with the peptide or protein. This increase coincides with a broad endothermic peak in the DSC, suggesting a disaggregation of both the peptide and the protein before the main phase transition of the lipid. Detailed simulations of the multicomponent ESR spectra have been performed by the latest nonlinear least-squares methods, which have helped to clarify the spectral interpretations. It is found that the simulations of ESR spectra from CSL in the gel phase for all the lipid membranes studied could be significantly improved by utilizing a model with CSL molecules existing as both hydrogen-bonded to the bilayer interface and non-hydrogen-bonded within the bilayer.
Deuterium magnetic resonance studies of the interaction of lipids with membrane proteins
Proceedings of the National Academy of Sciences, 1977
The deuterium magnetic resonance spectra of lipid-protein particles containing cytochrome c oxidase (ferrocytochrome c:oxygen oxidoreductase, EC 1.9.3.1) isolated from beef heart mitochondria and the specifically deuterated lipid 1416,16,16-trideuteropalmitoyl-2-palmitoleoyl phosphatidylcholine are presented. These reconstituted particles are of uniform lipid and protein content; however, the spectra clearly show two environments characterized by distinctly different residual quadrupolar splittings or order parameters. The lessordered environment shows a splitting similar to but slightly less than that of the pure lipid alone at a given temperature. The more restricted environment appears to be induced by the presence of the protein. The amount of the restricted lipid is clearly temperature dependent with a 2to 3fold decrease in relative amount from 2 to 220. The rate of exchange of lipid between the free and restricted environments is slower than 103/sec. The significance of these phenomena is discussed.
The protein–lipid interface: perspectives from magnetic resonance and crystal structures
Biochimica et Biophysica Acta (BBA) - Biomembranes, 2004
Lipid-protein interactions in membranes are dynamic, and consequently are well studied by magnetic resonance spectroscopy. More recently, lipids associated with integral membrane proteins have been resolved in crystals by X-ray diffraction, mostly at cryogenic temperatures. The conformation and chain ordering of lipids in crystals of integral proteins are reviewed here and are compared and contrasted with results from magnetic resonance and with the crystal structures of phospholipid bilayers. Various aspects of spin-label magnetic resonance studies on lipid interactions with single integral proteins are also reviewed: specificity for phosphatidylcholine, competition with local anaesthetics, oligomer formation of single transmembrane helices, and protein-linked lipid chains. Finally, the interactions between integral proteins and peripheral or lipid-linked proteins, as reflected by the lipid-protein interactions in double reconstitutions, are considered. D
Biochimica Et Biophysica Acta-biomembranes, 1989
Saturation transfer ESR has been used to study the dynamic behaviour of lipids in the appressed regions of thylakoid membranes from pea seedlings. Four different phospho- and galacto-lipid spin labels (phosphatidylcholine labelled at the 12 or 14 C-atom positions of the sn-2 chain, phosphatidylglycerol labelled at the 14-position of the sn-2 chain, and monogalactosyldiacylglycerol labelled at the 12-position of the sn-2 chain) were used to probe the lipid environment in photosystem II-enriched membranes prepared by detergent extraction. The ESR spectra show that the majority of the lipid in these preparations is strongly motionally restricted. Values for the effective rotational correlation times of the labelled chains were deduced from the lineheight ratios and integrals of thhe saturation transfer ESR spectra. The effective rotational correlation times were found to be in the 105 range, indicating a very low lipid chain mobility which correlates with the low lipid content of these preparations. Comparison of the effective rotational correlation times deduced from the different diagnostic regions of the spectrum revealed little anisotropy in the chain mobility, indicating that the dominant motional mode was trans-gauche isomerization. The effective rotational correlation times deduced from the spectral integrals were similar to those deduced from the lineheight ratios, consistent with the absence of any appreciable fluid lipid component in these preparations. The results also indicate some selectivity of interaction between the lipid species, with phosphatidylcholine exhibiting appreciably slower motion than either phosphatidylglycerol or monogalactosyldiacylglycerol.
Biochemistry, 1979
In order to fix spin-labeled acids at the boundary layer of membrane-bound proteins, spin-labeled long-chain derivatives (m,n)MSL (general formula, CH,(CH2),R-(CH2),COO(CH2)2-M, where R is an oxazolidine ring containing a nitroxide and M is a maleimide residue) were synthesized. The spin-labeled molecules bind covalently to at least two different classes of sulfhydryl groups on rhodopsin in disc membrane fragments from bovine retina. One class of sites is hydrophilic and corresponds to the two SH groups labeled readily by N-ethylmaleimide; the second class of sites is only reached by hydrophobic probes. (10,3)MSL binds equally well to the two classes of sites on rhodopsin, whereas (1 , I 4)MSL, more hydrophobic, binds preferentially to the hydrophobic sites. Apparently a third class of SH groups can be labeled if a very large excess of (m,n)MSL is employed, but proteins may be denatured in this latter case. Labels not covalently bound are removed from the membranes by incubation with fatty acid free bovine serum albumin. However, it is found that the probes do not bind only to rhodopsin in the disc membranes. (m,n)MSL also binds covalently to phosphatidylethanolamine in the rod outer segments or in liposomes. This covalent binding to phospholipids is demonstrated by lipid extraction and thin-layer chromatographic analysis. In order to obtain the pure EPR spectra of the spin-labeled fatty acids bound to the protein, the spectra corresponding to phospholipid-bound spin labels have been I t is often admitted that intrinsic membrane proteins are surrounded by a boundary layer or "annulus" of rigidly bound lipid. The immobilization of this shell of lipid has been deduced essentially from EPR experiments involving spin-labeled fatty acids incorporated into reconstituted systems containing variable lipid to protein ratios. were the first to propose from spin-label experiments the model of a boundary layer of lipid surrounding an intrinsic membrane protein, namely cytochrome oxidase. Later, Hesketh et al. (1976) reported similar experiments with Ca2+-ATPase, while Chapman et al. (1977) showed that gramcidin A can lead to the same EPR results, if this polypeptide is dissolved in a small amount of lipid.
Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels
Biochimica et Biophysica Acta (BBA) - Biomembranes, 1985
Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog red outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodopsin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.
Studies of Interactions Between Peptides/Proteins and Lipid
2010
Introduction of the bond additivity model 1.8.2. Symmetry point group of helical and antiparallel β-sheet structures 1.8.3. s and p polarized light 1.8.4. Fresnel coefficients 1.9 REFERENCES CHAPTER 2. ORIENTATION DETERMINATION OF PROTEIN HELICAL SECONDARY STRUCTURE USING LINEAR AND NONLINEAR VIBRATIONAL SPECTROSCOPY 2.1 INTRODUCTION vi 2.2 ORIENTATION DETERMINATION OF AN α-HELIX 2.2.1 Introduction of Pauling's α-helix 2.2.2 IR transition dipole moment of an α-helix amide I mode 2.2.3 Raman polarizability tensor of an α-helix amide I mode 2.2.4 SFG data analysis for α-helices based on the calculated IR transition dipole moment and Raman polarizability 2.2.5 The effect of varying the number of peptide units in an α-helical structure on SFG data analysis 2.2.6 Combination of measurements using different vibrational spectroscopic techniques 2.2.7 Discussion on the measurement of χ zzz with the near total reflection geometry 2.3 ORIENTATION DETERMINATION OF A 3-10 HELIX 2.3.1 IR transition dipole moment of a 3-10 helix amide I mode 2.3.2 Raman polarizability tensor of a 3-10 helix amide I mode 2.3.3 SFG data analysis for 3-10 helices based on the calculated IR transition dipole moment and Raman polarizability 2.4 CONCLUSION 2.5 REFERENCES CHAPTER 3. MOLECULAR INTERACTION BETWEEN MAGAININ 2 AND MODEL MEMBRANES IN SITU 3.1INTRODUCTION 3.2 MATERIALS AND METHODS 3.3 SFG DATA ANALYSIS 3.4 RESULTS AND DISCUSSIONS 3.4.1 SFG and ATR-FTIR Amide I Spectra 3.4.1.1 Magainin 2 in a POPG/POPG lipid bilayer 3.4.1.2 Magainin 2 in a POPC/POPC lipid bilayer 3.4.2. SFG spectra of POPG/POPG and POPC/POPC lipid bilayers 3.5 CONCLUSION 3.6 REFERENCES CHAPTER 4. SFG STUDY ON A MEMBRANE ANCHORED PROTEIN: CYTOCHROME B5 4.1 INTRODUCTION 4.2 MATERIALS AND EXPERIMENTAL PROCEDURES vii 4.3 ORIENTATION OF FULL LENGTH CYT B5 IN A dDMPC/dDMPC LIPID BILAYER 4.4 ORIENTATION OF MUTANT-CYT B5 (MCYT B5) IN THE dDMPC/dDMPC LIPID BILAYER 4.5 TIME DEPENDENT STUDIES ON THE INTERACTIONS OF THE FULL LENGTH CYT B5 AND THE MCYT B5 WITH THE dDMPC/dDMPC LIPID BILAYER 4.6 OBSERVING THE dDMPC/dDMPC LIPID BILAYER SIGNAL CHANGES CAUSED BY THE FULL LENGTH CYT B5 AND THE MCYT B5 4.7 EFFECTS OF THE LINKER LENGTH ON THE MCYT b5-LIPID BILAYER INTERACTIONS 4.8 TEMPERATURE DEPENDENT STUDY ON THE INSERTION OF MCYT B5'S ANCHORING TAIL INTO THE dDMPC/dDMPC LIPID BILAYER 4.9 STUDIES ON THE INTERACTIONS BETWEEN CYT B5/ MCYT B5 AND LIPID BILAYERS COMPOSED OF DIFFERENT LIPIDS OF VARIOUS CHAIN LENGTHS 4.10 CONCLUSION 4.11 REFERENCES CHAPTER 5. ORIENTATION DETERMINATION OF INTERFACIAL β-SHEET STRUCTURES IN SITU