Effects of a fungal lipase on membrane organization evaluated by fluorescence polarization

Abstract

Triglyceride lipase from Thermomyces lanuginosa (TlL) binds to the non-substrate small (40 nm) unilamelar vesicles of 1,2-dimiristoylglycero- sn-3-phosphoglycerol (DMPG-SUVs) in a catalytically active structure, whereas it adopts a catalytically incompetent form in binding to zwitterionic 1,2-dimiristoylglycero- sn-3-phosphocholine (DMPC-SUVs) or to large (100 nm) unilamelar DMPG (DMPG-LUVs) vesicles. Steady-state anisotropy measurements with probes that localize at different positions in the membrane give information on the effects of TlL (and its mutants) on the mobility of the phospholipids. All TlL mutants insert into the DMPG-SUVs and increase lipid order at the headgroup region and at the hydrophobic core of the lipid bilayer as well. The increase of the rigidity of the membrane that occurs in the gel and liquid crystal states, results in an increase of the phase transition temperature ( T m). Kinetic experiments with monolayers of 1,2-dicaprin demonstrate the thermal stability of the enzyme in the range of temperatures of the phase transition. Mutations in the tryptophan (Trp) residues of TlL reduce activity of this enzyme and affect its interaction with the membrane. The membrane insertions of TlL mutants with other than Trp substitutions are much more shallower and produce only small increases of T m, whereas mutation of lid-located Trp89 or mutation of any other Trp residue (117, 221, 260) result in a deeper penetration and significant increases of the T m. Lipid dynamics of DMPC-SUVs or DMPG-LUVs are not affected by any of the TlL mutants, despite their strong binding to the lipids revealed by resonance energy transfer (RET). These results are consistent with the lipase–lipid penetration model in which the “lid” region of TlL inserts into the highly curved anionic interface, thus stabilizing the “open” or active enzyme conformation, whereas TlL binds to the surface of zwitterionic and large (small curvature) anionic vesicles in a “closed” (or inactive) conformation, without insertion of the lid.