Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR

作者：Pu Mingming; Orr Andrew; Redfield Alfred G; Roberts Mary F^*

来源：JOURNAL OF BIOLOGICAL CHEMISTRY, 2010, 285(35): 26916-26922.

DOI：10.1074/jbc.M110.123083

Despite the profound physiological consequences associated with peripheral membrane protein localization, only a rudimentary understanding of the interactions of proteins with membrane surfaces exists because these questions are inaccessible by commonly used structural techniques. Here, we combine high resolution field-cycling (31)P NMR relaxation methods with spin-labeled proteins to delineate specific interactions of a bacterial phospholipase C with phospholipid vesicles. Unexpectedly, discrete binding sites for both a substrate analogue and a different phospholipid (phosphatidylcholine) known to activate the enzyme are observed. The lifetimes for the occupation of these sites (when the protein is anchored transiently to the membrane) are > 1-2 mu s (but < 1 ms), which represents the first estimate of an off-rate for a lipid dissociating from a specific site on the protein and returning to the bilayer. Furthermore, analyses of the spin-label induced NMR relaxation corroborates the presence of a discrete tyrosine-rich phosphatidylcholine binding site whose location is consistent with that suggested by modeling studies. The methodology illustrated here may

• 出版日期2010-8-27