Horváth L I, Brophy P J, Marsh D
Max-Planck Institut für Biophysikalische Chemie, Abteilung Spektroskopie, Göttingen, Germany.
Biophys J. 1993 Mar;64(3):622-31. doi: 10.1016/S0006-3495(93)81421-8.
The microwave saturation properties of various spin-labeled lipids in reconstituted complexes of the myelin proteolipid protein with dimyristoyl phosphatidylcholine have been studied both by conventional and saturation transfer electron spin resonance (ESR) spectroscopy. In the fluid phase, the conventional ESR spectra consist of a fluid and a motionally restricted (i.e., protein-associated) component, whose relative proportions can be determined by spectral subtractions and depend on the selectivity of the particular spin-labeled lipid for the protein. At 4 degrees C when the bulk lipid is in the gel phase, the integrated intensity of the saturation transfer ESR spectra displays a linear dependence on the fraction of motionally restricted lipid that is deduced from the conventional ESR spectra in the fluid phase, indicating the presence of distinct populations of free and protein-interacting lipid with no exchange between them on the saturation transfer ESR time scale in the gel phase. At 30 degrees C when the bulk lipid is in the fluid phase, the saturation transfer integral displays a nonlinear dependence on the fraction of motionally restricted lipid, consistent with exchange between the two lipid populations on the saturation transfer ESR time scale in the fluid phase. For lipid spin labels with different selectivities for the protein in complexes of fixed lipid/protein ratio, the data in the fluid phase are consistent with a constant (diffusion-controlled) on-rate for exchange at the lipid-protein interface. Values ranging between 1 and 9 x 10(6) s-1 are estimated for the intrinsic off-rates for exchange of spin-labeled stearic acid and phosphatidylcholine, respectively, at 30 degrees C. Conventional continuous wave saturation experiments lead to similar conclusions regarding the lipid exchange rates in the fluid and gel phases of the lipid/protein recombinants. The ESR saturation studies therefore demonstrate exchange on the time scale of the nitroxide spin-lattice relaxation at the lipid-protein interface of myelin proteolipid/dimyristoyl phosphatidylcholine complexes in the fluid phase but not in the gel phase.
利用传统的和饱和转移电子自旋共振（ESR）光谱，研究了髓鞘蛋白脂蛋白与二肉豆蔻酰磷脂酰胆碱重构复合物中各种自旋标记脂质的微波饱和特性。在液相中，传统的ESR光谱由一个流体成分和一个运动受限（即与蛋白质相关）的成分组成，其相对比例可通过光谱减法确定，并取决于特定自旋标记脂质对蛋白质的选择性。在4℃时，当总体脂质处于凝胶相时，饱和转移ESR光谱的积分强度与从液相中传统ESR光谱推导得出的运动受限脂质分数呈线性关系，表明在凝胶相中，存在自由脂质和与蛋白质相互作用脂质的不同群体，在饱和转移ESR时间尺度上它们之间没有交换。在30℃时，当总体脂质处于液相时，饱和转移积分与运动受限脂质分数呈非线性关系，这与液相中饱和转移ESR时间尺度上两个脂质群体之间的交换一致。对于固定脂质/蛋白质比例的复合物中对蛋白质具有不同选择性的脂质自旋标记，液相中的数据与脂质-蛋白质界面处交换的恒定（扩散控制）结合速率一致。在30℃时，自旋标记硬脂酸和磷脂酰胆碱交换的固有解离速率估计值分别在1至9×10⁶ s⁻¹之间。传统的连续波饱和实验对于脂质/蛋白质重组体的液相和凝胶相中的脂质交换速率得出了类似的结论。因此，ESR饱和研究表明，在髓鞘蛋白脂蛋白/二肉豆蔻酰磷脂酰胆碱复合物的脂质-蛋白质界面处，在液相中存在基于氮氧自由基自旋-晶格弛豫时间尺度的交换，但在凝胶相中不存在。
