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Interfacial orientation of Thermomyces lanuginosa lipase on phospholipid vesicles investigated by electron spin resonance relaxation spectroscopy
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 48, 14185-14196 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2002. Vol. 41, no 48, 14185-14196 p.
Keyword [en]
alpha/beta-hydrolase fold, candida-rugosa lipase, x-ray crystallography, humicola-lanuginosa, bacteriorhodopsin mutants, triacylglycerol lipase, conformational-changes, triglyceride lipase, substrate-binding, triad forms
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-22089DOI: 10.1021/bi020158rISI: 000179517000012OAI: oai:DiVA.org:kth-22089DiVA: diva2:340787
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2010-08-30Bibliographically approved
In thesis
1. Lipase-Lipid Interactions Studied by Site-Directed Labeling and Biological Spectroscopy
Open this publication in new window or tab >>Lipase-Lipid Interactions Studied by Site-Directed Labeling and Biological Spectroscopy
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

This thesis describes the study of lipase–lipid interactions of the triglyceride lipase Thermomyces lanuginosus lipase, TLL, by use of site-directed chemical labeling and biological spectroscopy. Several single-cysteine variants of TLL were produced, and labeled with probes containing either an affinity-, a fluorescence- or a spin-label functionality. A combined reduction- and labeling protocol was elaborated for the TLL single-cysteine variants, which were found to be prematurely oxidized during production. A diagnostic dot-blot method based on biotin-avidin interactions was established for the detection of free thiols in recombinant proteins. This method has a picomole detection limit and was found accurate in free thiol quantitation.

Unilamellar phospholipid vesicles consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG) or of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyl-choline (POPC) were produced as stable, well-defined lipid interfaces, to which TLL is known to bind with high affinity. The binding orientation of TLL was investigated, employing eleven spin-labeled TLL single-cysteine variants with the point-mutations positioned around the protein surface. Spin-relaxation enhancement was induced by the spin-relaxation agent chromium(III) oxalate (Crox), and a novel ESR-method for measuring the interactions of nitroxide spin-labels at low-microwave amplitudes was established. This method requires only standard ESR-instrumentation and is optimal for surface-exposed spin-labels. The interactions of TLL spin-labels with Crox in the aqueous phase were studied by ESR spectroscopy, while spin-label interactions with the lipid phase were analyzed by fluorescence quenching of dansyl-labeled vesicles. In addition, ESR-data was employed in conjunction with electrostatic potential-based modeling to dock TLL on the membrane of small POPG vesicles. This afforded information on the detailed molecular orientation of TLL at the lipid–water interface.

TLL was found to bind on 50-nm–diameter POPG vesicles in an activated form with the active-site entrance and the proximal part of the lid oriented against the lipid membrane. On 100 nm POPG vesicles and 50 nm POPC vesicles, TLL was oriented with the active site facing away from the membrane, corresponding to approximately a 180-degree–rotation about TLL’s vertical axis. There was no deep penetration of TLL residues in the lipid membranes, but TLL was found to associate closer to the negatively charged POPG-surface, than that of the zwitterionic POPC. TLL was also able to bind to two vesicles simultaneously, as revealed by fluorescence resonance energy transfer (FRET) between fluorescent labeled vesicles. The experimental data obtained provided insights into the interfacial behaviour of a triglyceride lipase.

Place, publisher, year, edition, pages
Stockholm: KTH, 2006. x, 68 p.
Keyword
thermomyces lanuginous lipase, site-directed labeling, lipase-lipid interactions
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-618 (URN)91-7178-253-2 (ISBN)
Public defence
2006-02-24, Svedbergssalen, AlbaNova universitetscentrum, Roslagstullsbacken 21, Stockholm, 13:00
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Note
QC 20100830Available from: 2006-02-10 Created: 2006-02-10 Last updated: 2010-08-30Bibliographically approved

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