| 1. |
- Hedin, Eva . M. K., et al.
(author)
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Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy
- 2002
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 41:48, s. 14185-14196
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Journal article (peer-reviewed)abstract
- 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 spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar 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 investigated using, a water-soluble spin relaxation agent. chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (< 0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure 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 quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results.
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| 2. |
- Hedin, Eva. M. K., et al.
(author)
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Selective reduction and chemical modification of oxidized lipase cysteine mutants
- 2002
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In: Canadian journal of chemistry (Print). - : Canadian Science Publishing. - 0008-4042 .- 1480-3291. ; 80:6, s. 529-539
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Journal article (peer-reviewed)abstract
- Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 3.1.1.3) Were produced and characterized for the purpose of site-directed chemical modification with spectroscopic reporter groups. All cysteine mutants were found to be predominantly blocked by oxidation to disulfides with endogenous cysteine during production. The fraction of lipase molecules with free sulfhydryl groups was analyzed by labeling with N-biotinylaminoethyl methanethiosulfonate, followed by a novel dot-blot method based on biotin-streptavidin interactions. A non-invasive method for the reduction of the introduced cysteine was elaborated for this protein containing three native disulfide bridges. The site-specifically reduced TLL mutants were then labeled with the sulfhydryl-specific reagents 2-(5-dimethylaminonaphth-1-ylsulfonamido)ethyl methanethiosulfonate or (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate, and studied by fluorescence and electron spin resonance (ESR) spectroscopy.
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