Spin-label ESR studies on the interaction of bovine spinal cord myelin basic protein with dimyristoylphosphatidylglycerol dispersions

Abstract

Electron spin resonance (ESR) spectroscopy and chemical binding assays were used to study the interaction of bovine spinal cord myelin basic protein (MBP) with dimyristoylphosphatidylglycerol (DMPG) membranes. Increasing binding of MBP to DMPG bilayers resulted in an increasing motional restriction of P G spin-labeled at the C-5 atom position in the acyl chain, up to a maximum degree of association of 1 MBP molecule per 36 lipid molecules. ESR spectra of PG spin-labels labeled at other positions in the sn-2 chain showed a similar motional restriction, while still preserving the chain flexibility gradient characteristic of fluid lipid bilayers. In addition, labels at the C-12 and C-14 atom positions gave twocomponent spectra, suggesting a partial hydrophobic penetration of the M B P into the bilayer. Spectral subtractions were used to quantitate the membrane penetration in terms of the stoichiometry of the lipid-protein complexes. Approximately 50% of the spin-labeled lipid chains were directly affected at saturation protein binding. The salt and pH dependence of the ESR spectra and of the protein binding demonstrated that electrostatic interaction of the basic residues of the MBP with the PG headgroups is necessary for an effective association of the MBP with phospholipid bilayers. Binding of the protein, and concomitant perturbation of the lipid chain mobility, was reduced as the ionic strength increased, until at salt concentrations above 1 M NaCl the protein was no longer bound. The binding and ESR spectral perturbation also decreased as the protein charge was reduced by pH titration to above the p l of the protein at approximately p H 10.

Selectivity of interaction of phospholipids with bovine spinal cord myelin basic protein studied by spin-label electron spin resonance

“…The saturation binding values of MBP to the DMPG/DMPC mixed bilayers are also given in Figure 2a. Consistent with earlier observations (Boggs & Moscarello, 1978b;Papahadjopoulos et al, 1975;Sankaram et al, 1989), it is seen from Figure 2a that the MBP binds strongly to DMPG bilayers with a lipid/protein ratio of 36:l mol/mol at saturation but does not bind appreciably to bilayers of DMPC alone. The dependence of the binding on mole fraction of DMPC, XDMp-, is not linear, indicating that the mode of protein binding to DMPG in the mixed bilayers is different from the binding to pure DMPG bilayers.…”

“…The slow exchange leads only to a slight broadening of this fluid component. The two-component nature of the spectra was substantiated and quantitated as described earlier (Marsh, 1982;Gorrissen et al, 1986;Sankaram et al, 1989). Briefly, the method involves subtracting a fluid lipid spectrum obtained for the spin-label in pure lipid bilayers from the spectrum of the lipid-protein recombinant.…”

“…Sample temperatures were controlled by a nitrogen gas flow regulation system and were measured by using a fine-wire thermocouple that was placed close to the sample at the top of the microwave cavity. Spectral subtractions were performed as described earlier (Marsh, 1982;Sankaram et al, 1989). The outer hyperfine splitting, 2A, , was used to characterize the spectra of the C-5 spin-labels.…”

“…The MBP has no detectable tertiary structure in aqueous solutions (Eylar & Thompson, 1969;Chao & Einstein, 1970;Palmer & Dawson, 1969) but adopts a-helical and &sheet conformations in nonpolar environments like organic solvent mixtures (Liebes et al, 1975;Stone et al, 1985), micelles (Mendz et al, 1984), and lipid bilayers (Surewicz et al, 1987). A strong binding to acidic lipids (Palmer & Dawson, 1969) static in nature, hydrophobic interactions that presumably arise from a partial penetration of the protein into membranes have been detected by using differential scanning calorimetry (Papahadjopoulos et al, 1975), nuclear magnetic resonance (Mendz et al, 1984), and spin-label electron spin resonance (Boggs & Moscarello, 1978b;Sankaram et al, 1989) spectroscopic techniques.…”

Cytochrome c-Induced Increase of Motionally Restricted Lipid in Reconstituted Cytochrome c Oxidase Membranes, Revealed by Spin-Label ESR Spectroscopy

“…The data for the myelin proteins was interpreted in terms of a mutual steric exclusion model in which the boundary layer of lipid surrounding the proteolipid protein is excluded from interaction with the basic protein, and the latter binds only to the bulk (i.e., nonboundary) lipid surface. Both proteolipid and myelin basic proteins exhibit a strong selectivity for negatively charged lipids (28,29). Our finding that cytochrome c binds even to the lipids adjacent to cytochrome c oxidase (i.e., no steric exclusion) suggests a rather strong electrostatic binding of cytochrome c to negatively charged lipids such as DMPG.…”