Selective reduction and chemical modification of oxidized lipase cysteine mutants
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
Thirteen single-cysteine mutants of the 33 kDa fungal triacylglycerol lipase Thermomyces (formerly Humicola) lanuginosa lipase (TLL, EC 126.96.36.199) 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.
lipase, cysteine mutant, selective reduction, chemical modification, methanethiosulfonate, humicola-lanuginosa lipase, fluorescence spectroscopy, conformational-changes, protein, stability, sulfhydryl, dynamics, enzymes, binding, thiols
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
IdentifiersURN: urn:nbn:se:kth:diva-21725DOI: 10.1139/v02-046ISI: 000176891000003OAI: oai:DiVA.org:kth-21725DiVA: diva2:340423
QC 201005252010-08-102010-08-102010-08-30Bibliographically approved