Surface accessibility of 13C-labeled lysine residues in membrane-bound myelin basic protein.
Surface-exposed regions of membrane-bound myelin basic protein--the major extrinsic membrane protein of central nervous system myelin--have been implicated as possible antigenic sites in diseased myelin. With the goal of determining the extent and nature of these regions, we have prepared basic protein modified with 13CH3-enriched acetyl groups at 7 of its 13 lysine residues. The resulting protein was placed in a membrane environment and studied by NMR spectroscopy to determine the location and rates of molecular motion of the labeled side chains with respect to lipid bilayers of the membrane. When 13C NMR spectra were obtained of the acetylated protein bound to multilamellar vesicles prepared from dimyristoylphosphatidic acid in the gel state (T = 33 degrees C), conditions under which reduced motion in the lipid bilayer broadens methylene and methyl 13C resonances of the membrane beyond detection (i.e. greater than 75-100 Hz), line widths of membrane-bound protein were measured to be 7.8 Hz, an increase of 4 Hz versus free protein. A reduction of 25-30% in integrated intensity observed in protein acetyl resonances upon membrane interaction was shown to be attributable to a population of protein-aggregated liposomes whose resonances were similarly too broad to be observed. Thus, the epsilon-acetyllysyl probes distributed throughout the protein do not penetrate the dimyristoylphosphatidic acid bilayer, but must reside in the interstitial aqueous spaces at or between membrane surfaces. These findings suggest an overall surface accessibility of membrane-bound myelin basic protein and are therefore incompatible with a model for the protein involving membrane-embedded loops or regions of functional significance.
D000089:Acetic Anhydrides; D000818:Animals; D001921:Brain / Q000378:metabolism; D002250:Carbon Radioisotopes; D002417:Cattle; D004134:Dimyristoylphosphatidylcholine; D008081:Liposomes; D008239:Lysine; D009682:Magnetic Resonance Spectroscopy; D004676:Myelin Basic Protein / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D011663:Pulmonary Surfactants
D000089:Acetic Anhydrides; D002250:Carbon Radioisotopes; D008081:Liposomes; D004676:Myelin Basic Protein; D011663:Pulmonary Surfactants; C031800:acetic anhydride; D008239:Lysine; D004134:Dimyristoylphosphatidylcholine