| Abstract
| - The triglyceride lipase (EC 3.1.1.3) Thermomyces lanuginosus lipase (TLL) binds with highaffinity to unilamellar phospholipid vesicles that serve as a diluent interface for both lipase and substrate,but it displays interfacial activation on only small and negatively charged such vesicles [Cajal, Y., et al.(2000) Biochemistry 39, 413−423]. The productive-mode binding orientation of TLL at the lipid−waterinterface of small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) was previously determined using electron spin resonance (ESR) spectroscopy incombination with site-directed spin-labeling [Hedin, E. M. K., et al. (2002) Biochemistry 41, 14185−14196]. In our investigation, we have studied the interfacial orientation of TLL when bound to largeunilamellar vesicles (LUV) consisting of POPG, and bound to SUV consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC). Eleven single-cysteine TLL mutants were spin-labeled aspreviously described, and studied upon membrane binding using the water soluble spin-relaxation agentchromium(III) oxalate (Crox). Furthermore, dansyl-labeled vesicles revealed the intermolecular fluorescencequenching efficiency between each spin-label positioned on TLL, and the lipid membrane. ESR exposureand fluorescence quenching data show that TLL associates closer to the negatively charged PG surfacethan the zwitterionic PC surface, and binds to both POPG LUV and POPC SUV predominantly throughthe concave backside of TLL opposite the active site, as revealed by the contact residues K74C-SL, R209C-SL, and T192C-SL. This orientation is significantly different compared to that on the POPG SUV, andmight explain the differences in activation of the lipase. Evidently, both the charge and accessibility(curvature) of the vesicle surface determine the TLL orientation at the phospholipid interface.
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