Defining specific lipid binding sites for a peripheral membrane protein in situ using subtesla field-cycling NMR.
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 micros (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.
Pu
Mingming
M
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02465.
Orr
Andrew
A
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02465.
Redfield
Alfred G
AG
Department of Biochemistry, Brandeis University, Waltham, Massachusetts 024547.
Roberts
Mary F
MF
Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02465. Electronic address: mary.roberts@bc.edu.
D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D008051:Lipid Bilayers / Q000737:chemistry; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D010738:Type C Phospholipases / Q000737:chemistry* / Q000235:genetics
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D010738:Type C Phospholipases