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Selective reduction and chemical modification of oxidized lipase cysteine mutants
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
KTH, Superseded Departments, Biotechnology.
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2002 (English)In: Canadian journal of chemistry (Print), ISSN 0008-4042, E-ISSN 1480-3291, Vol. 80, no 6, 529-539 p.Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
2002. Vol. 80, no 6, 529-539 p.
Keyword [en]
lipase, cysteine mutant, selective reduction, chemical modification, methanethiosulfonate, humicola-lanuginosa lipase, fluorescence spectroscopy, conformational-changes, protein, stability, sulfhydryl, dynamics, enzymes, binding, thiols
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
URN: urn:nbn:se:kth:diva-21725DOI: 10.1139/v02-046ISI: 000176891000003OAI: oai:DiVA.org:kth-21725DiVA: diva2:340423
Note
QC 20100525Available from: 2010-08-10 Created: 2010-08-10 Last updated: 2017-12-12Bibliographically 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
Opponent
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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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Berglund, Per

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