Interfacial Orientation of Thermomyces lanuginosa Lipase on Phospholipid Vesicles Investigated by Electron Spin Resonance Relaxation Spectroscopy

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• 作者：Eva M. K. Hedin ; Pernille H//pubs.acs.org/images/gifchars/oslash.gif" border="0">yrup ; Shamkant A. Patkar ; Jesper Vind ; Allan Svendsen ; Linda Fransson ; and Karl Hult
• 刊名：Biochemistry
• 出版年：2002
• 出版时间：December 3, 2002
• 年：2002
• 卷：41
• 期：48
• 页码：14185 - 14196
• 全文大小：463K
• 年卷期：v.41,no.48(December 3, 2002)
• ISSN：1520-4995

文摘

The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL,EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spinresonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutationpositioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-³-pyrroline-3-methyl) methanethiosulfonate. These were studied together with smallunilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG),to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000)Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigatedusing a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESRrelaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwaveamplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface ofTLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys andThr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowestexposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface.As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenchingof dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmerquenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientationof TLL is proposed on the basis of the obtained results.