Altenbach C, Froncisz W, Hyde J S, Hubbell W L
Jules Stein Eye Institute, University of California, Los Angeles 90024-1771.
Biophys J. 1989 Dec;56(6):1183-91. doi: 10.1016/S0006-3495(89)82765-1.
Melittin spin-labeled specifically with a nitroxide at positions 7, 21, 23, or the amino terminus was bound to phospholipid membranes, and the exposure of the spin label to the aqueous phase was investigated by measurement of Heisenberg exchange with chromium oxalate in the solution. The exchange frequency was determined by saturation recovery electron paramagnetic resonance (EPR) using a loop-gap resonator. This method allows use of very low concentrations (less than 1 mM) of chromium oxalate compared with conventional measurements of EPR line broadening (typically 50 mM), thus avoiding problems associated with high metal ion concentration. Differences in exchange frequency between the various positions were also estimated by continuous wave power saturation methods. In either approach, the spin label at lysine 7 was found to be the most exposed to chromium oxalate whereas that at lysine 23 was found to be the least exposed. This is consistent with a model for the membrane bound peptide in which an amphiphilic helix lies with its axis parallel to the bilayer surface and the hydrophobic moment points toward the bilayer interior.
将在第7、21、23位或氨基末端特异性标记有氮氧化物的蜂毒肽与磷脂膜结合，并通过测量其与溶液中草酸铬的海森堡交换来研究自旋标记物向水相的暴露情况。交换频率通过使用环形间隙谐振器的饱和恢复电子顺磁共振（EPR）来确定。与传统的EPR线宽测量（通常为50 mM）相比，该方法允许使用极低浓度（小于1 mM）的草酸铬，从而避免了与高金属离子浓度相关的问题。不同位置之间的交换频率差异也通过连续波功率饱和方法进行了估计。在这两种方法中，发现赖氨酸7处的自旋标记物对草酸铬的暴露程度最高，而赖氨酸23处的自旋标记物对草酸铬的暴露程度最低。这与膜结合肽的模型一致，在该模型中，两亲性螺旋的轴与双层表面平行，疏水矩指向双层内部。
