Magnetic resonance of membranes

Knowles, Peter F.; Marsh, D.

doi:10.1042/bj2740625

Cited by 69 publications

(41 citation statements)

References 222 publications

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“…This implies that the peptide must be integrated in the bilayer in a polymeric P-sheet form consisting of many more strands than a single peptide monomer [cf. Marsh and Horvhth (1989) and Knowles and Marsh (1991)l. Such a structure, e.g., a @-barrel, would completely satisfy the H-bonding capacity of the peptide backbone, as is required on energetic grounds for a peptide embedded in the hydrophobic region of a membrane.…”

Section: Discussionmentioning

confidence: 99%

Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

Horváth¹,

Heimburg²,

Kovachev³

et al. 1995

Biochemistry

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The 26-residue peptide of sequence KEALYILMVLGFFGFFTLGILSYIR, which contains the single putative transmembrane domain of a small protein that is associated with slow voltage-gated K+ channels, has been incorporated in bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol to form complexes of homogeneous lipidpeptide ratio. Fourier transform infrared spectroscopy indicates that the peptide is integrated in the lipid bilayer wholly in a @-sheet conformation. The electron spin resonance spectra of spin-labeled lipids in the lipidpeptide complexes contain a component corresponding to lipids whose chains are motionally restricted in a manner similar to those of lipids at the hydrophobic surface of integral transmembrane proteins. From the dependence of the lipid spin label spectra on the lipidpeptide ratio of the complexes, it is found that ca. 2.5 lipids per peptide monomer, independent of the species of spin-labeled lipid, are motionally restricted by direct interaction with the peptide in the bilayer. This value would be consistent with, e.g., a @-barrel structure for the peptide in which the @-strands either are strongly tilted or have a reverse turn at their center. A preferential selectivity of interaction with the peptide is observed for the negatively charged spin-labeled lipids phosphatidic acid, stearic acid, and phosphatidylserine, which indicates close proximity of the positively charged residues at the peptide termini to the lipid headgroups. The saturation-transfer electron spin resonance spectra of the peptide spin-labeled at a cysteine residue replacing Leu18 evidence rather slow rotational diffusion in the lipid complexes. This indicates that the presumably enclosed @-sheet units of the peptide are aggregated in oligomeric assemblies in the lipid bilayer. The results suggest a way in which one type of channel unit may be integrated in the membrane.Ion channels are generally large, often multimeric, proteins containing many transmembrane segments (Stephenson, 1991). In spite of the molecular complexity, it is likely that the channel itself is lined with a limited range of, possibly homologous, transmembrane segments. Correlating with thls, it is found that certain relatively short synthetic peptides, of sequences resembling those of putative pore-lining segments, are able to sustain channel activity when incorporated in lipid bilayers (Montal, 1990;Ben-Efraim et al., 1993). Such synthetic peptides are therefore suitable models for studying particular aspects of channel structure and assembly by using biophysical or biochemical techniques. The direct relevance to native channels depends, of course, on the correct identification of the pore-lining transmembrane segments.In addition to the complex channel proteins mentioned above, the DNA clone encoding a small protein associated with a slowly activating voltage-gated potassium channel has been identified in rat kidney (Takumi et al., 1988). This protein, Z s~, contains only 130 amino acids with a single putative 23-residu...

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Section: Discussionmentioning

confidence: 99%

Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

Horváth¹,

Heimburg²,

Kovachev³

et al. 1995

Biochemistry

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“…The implications of these two different forms of insertion of SP-B in phosphatidylcholine bilayers for the lipid-protein interactions have been analyzed by electron spin resonance (ESR) spectroscopy. This technique can give detailed information about the effect of the protein on the thermotropic behavior of the phospholipids, and also positional resolution of the direct motional restriction of the lipid chains upon their interaction with the protein (13,31). The use of phospholipid probes with nitroxide spin-labels at different positions along the acyl chain in previous studies showed that SP-B increases the order and decreases the mobility of acyl chains in phosphatidylcholine bilayers in the fluid phase, without affecting the gel phase, at least on the time scale of conventional ESR (13).…”

Section: Insertion Of Sp-b In Pc Bilayersmentioning

confidence: 99%

Depth Profiles of Pulmonary Surfactant Protein B in Phosphatidylcholine Bilayers, Studied by Fluorescence and Electron Spin Resonance Spectroscopy

et al. 1998

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Pulmonary surfactant-associated protein B (SP-B) has been isolated from porcine lungs and reconstituted in bilayers of dipalmitoylphosphatidylcholine (DPPC) or egg yolk phosphatidylcholine (PC) to characterize the extent of insertion of the protein into phospholipid bilayers. The parameters for the interaction of SP-B with DPPC or PC using different reconstitution protocols have been estimated from the changes induced in the fluorescence emission spectrum of the single protein tryptophan. All the different reconstituted SP-B-phospholipid preparations studied had similar K d values for the binding of the protein to the lipids, on the order of a few micromolar. The depth of penetration of SP-B into phospholipid bilayers has been estimated by the parallax method, which compares the relative efficiencies of quenching of the protein fluorescence by a shallow or a deeper spin-labeled phospholipid probe. SP-B tryptophan was found to be located 10-13 Å from the center of bilayers, which is consistent with a superficial location of SP-B in phosphatidylcholine membranes. Parallax experiments, as well as resonance energy transfer from SP-B tryptophan to an acceptor probe located in the center of the bilayer, indicate that there are significant differences in the extent of insertion of the protein, depending on the method of reconstitution. SP-B reconstituted from lipid/protein mixtures in organic solvents is inserted more deeply in PC or DPPC bilayers than the protein reconstituted by addition to preformed phospholipid vesicles. These differences in the extent of insertion lead to qualitative and quantitative differences in the effect of the protein on the mobility of the phospholipid acyl chains, as studied by spin-label electron spin resonance (ESR) spectroscopy, and could represent different functional stages in the surfactant cycle.

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“…The 7 R was equal to 1.55 x 10"O s (Table 3), as determined from Equation l . The isotropic hyperfine coupling constant (ah = 15.34 G) was also measured (Knowles et al, 1976). B: SP-Bf-,, samples two distinct environments in PBS.…”

Section: Lm Gordon Et Almentioning

confidence: 99%

Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments

et al. 1996

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Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1_25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B,-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B,_,, sequence. Residues Cys-8-Gly-25 are largely a-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-Bl-25 spin-labeled at the N-terminal Phe (i.e., SP-BT_25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-BI-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-BI-2S interacting with lipids.

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Magnetic resonance of membranes

Cited by 69 publications

References 222 publications

Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

Integration of a K+ Channel-Associated Peptide in a Lipid Bilayer: Conformation, Lipid-Protein Interactions, and Rotational Diffusion

Depth Profiles of Pulmonary Surfactant Protein B in Phosphatidylcholine Bilayers, Studied by Fluorescence and Electron Spin Resonance Spectroscopy

Conformation and molecular topography of the N‐terminal segment of surfactant protein B in structure‐promoting environments

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