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How does Bacillus thuringiensis PI‐Phospholipase C bind to mixed component vesicles? Insights from mass spectrometry H‐D exchange
Author(s) -
Shi Xiaomeng,
Roberts Mary F.,
Gershenson Anne,
Meklemburg Robert,
Wintrode Patrick L.
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.520.12
Subject(s) - chemistry , phosphatidylglycerol , vesicle , membrane , dimer , phosphatidylcholine , halobacterium salinarum , biophysics , helix (gastropod) , biochemistry , phospholipid , bacteriorhodopsin , biology , ecology , organic chemistry , snail
It is difficult to determine the orientation and conformation of peripheral membrane proteins when docked to target membranes. Often, flexible regions of the proteins provide the major contacts with the membranes. B. thuringiensis PI‐PLC has two discrete binding sites for phospholipids ‐ the active site binding PI (or substrate competitors such as phosphatidylglycerol (PG)), and an activator site specific for phosphatidylcholine (PC). We have performed H/D exchange mass spectrometry (HDXMS) experiments on PI‐PLC in solution or when tightly bound to 1:1 and 1:9 PG/PC small unilamellar vesicles. In the absence of vesicles, most regions of the protein exchange rapidly. However, with SUVs, the regions around helix B and helix C show reduced exchange, consistent with protection by the membrane. These results agree with mutagenesis studies identifying residues on those helices as important for membrane binding. Other significant changes include reduced exchange in β‐strand E and dramatically increased exchange in helix E. The H/D exchange rates were also determined for a constructed covalent dimer (disulfide‐linked W242C), thought to mimic the membrane‐induced dimeric structure but in the absence of membranes, and a mutant protein, W47A/W242A, with very low affinity for vesicles that exists as a dimer in solution. Results provide insight into self‐association of PI‐PLC monomers. Clearly, for peripheral membrane protein dynamics, HDXMS can provide information on structural changes not accessible by other methods (Supported by N.I.H. GM60418).