Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function

Jakob Skjold-Jørgensen; Jesper Vind; Olga V. Moroz; Elena Blagova; Vikram Kjøller Bhatia; Allan Svendsen; Keith S. Wilson; Morten Jannik Bjerrum

doi:10.1016/j.bbapap.2016.09.016

Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function

Jakob Skjold-Jørgensen, Jesper Vind, Olga V. Moroz, Elena Blagova, Vikram Kjøller Bhatia, Allan Svendsen, Keith S. Wilson, Morten Jannik Bjerrum

Kemisk Institut

20 Citationer (Scopus)

Abstract

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~ 10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions

Originalsprog	Engelsk
Tidsskrift	B B A - Proteins and Proteomics
Vol/bind	1865
Udgave nummer	1
Sider (fra-til)	20-27
Antal sider	8
ISSN	1570-9639
DOI	https://doi.org/10.1016/j.bbapap.2016.09.016
Status	Udgivet - 1 jan. 2017

Adgang til dokumentet

10.1016/j.bbapap.2016.09.016

Citationsformater

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title = "Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function",

abstract = "Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~ 10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions",

keywords = "TlL, Thermomyces lanuginosus lipase, QCM-D, quartz crystal microbalance with dissipation monitoring, Intrinsic switch, Interfacial activation, Lid, Interfacial binding, Crystal structure",

author = "Jakob Skjold-J{\o}rgensen and Jesper Vind and Moroz, {Olga V.} and Elena Blagova and Bhatia, {Vikram Kj{\o}ller} and Allan Svendsen and Wilson, {Keith S.} and Bjerrum, {Morten Jannik}",

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T1 - Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function

AU - Skjold-Jørgensen, Jakob

AU - Vind, Jesper

AU - Moroz, Olga V.

AU - Blagova, Elena

AU - Bhatia, Vikram Kjøller

AU - Svendsen, Allan

AU - Wilson, Keith S.

AU - Bjerrum, Morten Jannik

PY - 2017/1/1

Y1 - 2017/1/1

N2 - Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~ 10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions

AB - Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~ 10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions

KW - TlL, Thermomyces lanuginosus lipase

KW - QCM-D, quartz crystal microbalance with dissipation monitoring

KW - Intrinsic switch

KW - Interfacial activation

KW - Lid

KW - Interfacial binding

KW - Crystal structure

U2 - 10.1016/j.bbapap.2016.09.016

DO - 10.1016/j.bbapap.2016.09.016

M3 - Journal article

C2 - 27693248

SN - 1570-9639

VL - 1865

SP - 20

EP - 27

JO - B B A - Proteins and Proteomics

JF - B B A - Proteins and Proteomics

IS - 1

ER -