Selective reduction and chemical modification of oxidized lipase cysteine mutants

QC 20100525
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.

MEDICIN OCH HÄLSOVETENSKAP Medicinsk bioteknologi Medicinsk bioteknologi hsv//swe
MEDICAL AND HEALTH SCIENCES Medical Biotechnology Medical Biotechnology hsv//eng
lipase
cysteine mutant
selective reduction
chemical modification
methanethiosulfonate
humicola-lanuginosa lipase
fluorescence spectroscopy
conformational-changes
protein
stability
sulfhydryl
dynamics
enzymes
binding
thiols
Biochemistry
Biokemi

Patkar, S. A.KTH,Bioteknologi (författare)
Vind, J.KTH,Bioteknologi (författare)
Svendsen, A.KTH,Bioteknologi (författare)
Hult, KarlKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1nzlwuj (författare)
Berglund, PerKTH,Biokemi och biokemisk teknologi(Swepub:kth)u1271chh (författare)
KTHBioteknologi (creator_code:org_t)

Ingår i:Canadian journal of chemistry (Print): Canadian Science Publishing80:6, s. 529-5390008-40421480-3291

Av författaren/redakt...: Hedin, Eva. M. K ...; Patkar, S. A.; Vind, J.; Svendsen, A.; Hult, Karl; Berglund, Per

Om ämnet

MEDICIN OCH HÄLSOVETENSKAP: MEDICIN OCH HÄLS ...; och Medicinsk biotek ...; och Medicinsk biotek ...

Kungliga biblioteket hanterar dina personuppgifter i enlighet med EU:s dataskyddsförordning (2018), GDPR. Läs mer om hur det funkar här.
Så här hanterar KB dina uppgifter vid användning av denna tjänst.