Defining Specific Lipid Binding Sites for a Peripheral Membrane Protein in Situ Using Subtesla Field-cycling NMR |
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Authors: | Mingming Pu Andrew Orr Alfred G. Redfield Mary F. Roberts |
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Affiliation: | From the ‡Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02465 and ;the §Department of Biochemistry, Brandeis University, Waltham, Massachusetts 024547 |
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Abstract: | 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 31P 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 μs (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 be extended to a wide range of peripheral membrane proteins. |
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Keywords: | Membrane Bilayer Membrane Enzymes NMR Phosphatidylcholine Phospholipase C Phospholipid Vesicle |
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