Kinetics of Binding of Fluorescent Ligands to Enzymes with
Engineered Access Tunnels

J 2017

Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels

KAUSHIK, Shubhangi, Zbyněk PROKOP, Jiří DAMBORSKÝ a Radka CHALOUPKOVÁ

Základní údaje

Originální název

Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels

Autoři

KAUSHIK, Shubhangi (356 Indie, domácí), Zbyněk PROKOP (203 Česká republika, domácí), Jiří DAMBORSKÝ (203 Česká republika, garant, domácí) a Radka CHALOUPKOVÁ (203 Česká republika, domácí)

Vydání

FEBS Journal, Hoboken, NJ USA, WILEY-BLACKWELL, 2017, 1742-464X

Další údaje

Jazyk

angličtina

Typ výsledku

Článek v odborném periodiku

Obor

10608 Biochemistry and molecular biology

Stát vydavatele

Spojené státy

Utajení

není předmětem státního či obchodního tajemství

Odkazy

URL

Impakt faktor

Impact factor: 4.530

Kód RIV

RIV/00216224:14310/17:00095411

Organizační jednotka

Přírodovědecká fakulta

DOI

http://dx.doi.org/10.1111/febs.13957

UT WoS

000393601200010

Klíčová slova anglicky

binding kinetics; fluorescence polarization; haloalkane dehalogenases; HaloTag ligands; modified access tunnels

Štítky

NZ, rivok

Změněno: 5. 4. 2018 22:46, Ing. Nicole Zrilić

Anotace

V originále

Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutation introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method’s application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as alfa/beta-hydrolases containing >100,000 protein sequences based on the Pfam database.

Návaznosti

EE2.3.30.0037, projekt VaV

Název: Zaměstnáním nejlepších mladých vědců k rozvoji mezinárodní spolupráce

GAP207/12/0775, projekt VaV

Název: Strukturně-funkční vztahy haloalkan dehalogenas

Investor: Grantová agentura ČR, Structure-functional Relationships of Haloalkane Dehalogenases

LH14027, projekt VaV

Název: Nové koncepty a nástroje pro racionální design enzymů

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Nové koncepty a nástroje pro racionální design enzymů

LO1214, projekt VaV

Název: Centrum pro výzkum toxických látek v prostředí (Akronym: RECETOX)

Investor: Ministerstvo školství, mládeže a tělovýchovy ČR, Centrum pro výzkum toxických látek v prostředí

MUNI/M/1888/2014, interní kód MU

Název: Pokročilé hybridní metody studia transportních procesů v proteinech a jejich využití v designu biokatalyzátorů

Investor: Masarykova univerzita, Pokročilé hybridní metody studia transportních procesů v proteinech a jejich využití v designu biokatalyzátorů, INTERDISCIPLINARY - Mezioborové výzkumné projekty

Citovat

KAUSHIK, Shubhangi, Zbyněk PROKOP, Jiří DAMBORSKÝ a Radka CHALOUPKOVÁ. Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels. FEBS Journal. Hoboken, NJ USA: WILEY-BLACKWELL, 2017, roč. 284, č. 1, s. 134-148. ISSN 1742-464X. Dostupné z: https://dx.doi.org/10.1111/febs.13957.

@article{1403852,
   author = {Kaushik, Shubhangi and Prokop, Zbyněk and Damborský, Jiří and Chaloupková, Radka},
   article_location = {Hoboken, NJ USA},
   article_number = {1},
   doi = {http://dx.doi.org/10.1111/febs.13957},
   keywords = {binding kinetics; fluorescence polarization; haloalkane dehalogenases; HaloTag ligands; modified access tunnels},
   language = {eng},
   issn = {1742-464X},
   journal = {FEBS Journal},
   title = {Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels},
   url = {https://loschmidt.chemi.muni.cz/peg/publications/kinetics-of-binding-of-fluorescent-ligands-to-enzymes-with-engineered-access-tunnels/},
   volume = {284},
   year = {2017}
}

TY  - JOUR
ID  - 1403852
AU  - Kaushik, Shubhangi - Prokop, Zbyněk - Damborský, Jiří - Chaloupková, Radka
PY  - 2017
TI  - Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels
JF  - FEBS Journal
VL  - 284
IS  - 1
SP  - 134-148
EP  - 134-148
PB  - WILEY-BLACKWELL
SN  - 1742464X
KW  - binding kinetics
KW  - fluorescence polarization
KW  - haloalkane dehalogenases
KW  - HaloTag ligands
KW  - modified access tunnels
UR  - https://loschmidt.chemi.muni.cz/peg/publications/kinetics-of-binding-of-fluorescent-ligands-to-enzymes-with-engineered-access-tunnels/
N2  - Molecular recognition mechanisms and kinetics of binding of ligands to buried active sites via access tunnels are not well understood. Fluorescence polarization enables rapid and non-destructive real-time quantification of the association between small fluorescent ligands and large biomolecules. In this study, we describe analysis of binding kinetics of fluorescent ligands resembling linear halogenated alkanes to haloalkane dehalogenases. Dehalogenases possess buried active sites connected to the surrounding solvent by access tunnels. Modification of these tunnels by mutagenesis has emerged as a novel strategy to tailor the enzyme properties. We demonstrate that the fluorescence polarization method can sense differences in binding kinetics originating from even single mutation introduced to the tunnels. The results show, strikingly, that the rate constant of the dehalogenase variants varied across seven orders of magnitude and the type of ligand used strongly affected the binding kinetics of the enzyme. Furthermore, fluorescence polarization could be applied to cell-free extracts instead of purified proteins, extending the method’s application to medium-throughput screening of enzyme variant libraries generated in directed evolution experiments. The method can also provide in-depth kinetic information about the rate-determining step in binding kinetics and reveals the bottlenecks of enzyme accessibility. Assuming availability of appropriate fluorescent ligand, the method could be applied for analysis of accessibility of tunnels and buried active sites of enzymes forming a covalent alkyl-enzyme intermediate during their catalytic cycle, such as alfa/beta-hydrolases containing >100,000 protein sequences based on the Pfam database.
ER  -

KAUSHIK, Shubhangi, Zbyněk PROKOP, Jiří DAMBORSKÝ a Radka CHALOUPKOVÁ. Kinetics of Binding of Fluorescent Ligands to Enzymes with Engineered Access Tunnels. \textit{FEBS Journal}. Hoboken, NJ USA: WILEY-BLACKWELL, 2017, roč.~284, č.~1, s.~134-148. ISSN~1742-464X. Dostupné z: https://dx.doi.org/10.1111/febs.13957.

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