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High Resolution Field Cycling NMR Relaxometry Identifies Novel Phospholipid Binding Sites on PTEN and the Akt1 PH Domain
Author(s) -
Roberts Mary,
Stec Boguslaw,
Gradziel Cheryl,
Wei Yang,
Redfield Alfred
Publication year - 2015
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.29.1_supplement.886.17
Subject(s) - phospholipid , chemistry , biophysics , lipid bilayer , binding site , protein–lipid interaction , popc , c2 domain , membrane , biochemistry , membrane protein , integral membrane protein , biology
Amphitropic proteins have a variety of strategies to bind to target membranes. Electrostatic interactions often drive them to the membrane where other interactions occur. The latter can include hydrophobic side chain insertion and binding of specific phospholipid headgroups as well as phospholipid cation – protein aromatic pi interactions that transiently anchor the protein to the membrane. However, it is often hard to identify and characterize specific phospholipid binding sites on the protein. We have developed high resolution 31 P field cycling relaxometry, where the spin‐lattice relaxation rate is measured over a wide range of magnetic fields (typically 11.7 down to 0.004 T) as a method to define such phospholipid binding sites in terms of residence times on the protein and to obtain distances of the bound amphiphile to one or more spin‐labels on the target protein. When a crystal structure of the protein is available, the distances can then be used to search the surface for these discrete phospholipid binding sites. Use of the technique is illustrated with two examples: (i) the Akt1 PH domain interacting with cytotoxic alkylphospholipids and 3‐deoxy‐phosphatidylinositols, and (ii) PTEN binding its product and activator phosphatidylinositol 4,5‐bisphosphate in a site distinct form the active site. In both cases, the new site detected sheds light on how that particular lipid achieves it biological effect. This work was supported by National Science Foundation Grant MCB‐0959331.