J 2017

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

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

Basic information

Original name

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

Authors

KAUSHIK, Shubhangi (356 India, belonging to the institution), Zbyněk PROKOP (203 Czech Republic, belonging to the institution), Jiří DAMBORSKÝ (203 Czech Republic, guarantor, belonging to the institution) and Radka CHALOUPKOVÁ (203 Czech Republic, belonging to the institution)

Edition

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

Other information

Language

English

Type of outcome

Článek v odborném periodiku

Field of Study

10608 Biochemistry and molecular biology

Country of publisher

United States of America

Confidentiality degree

není předmětem státního či obchodního tajemství

References:

Impact factor

Impact factor: 4.530

RIV identification code

RIV/00216224:14310/17:00095411

Organization unit

Faculty of Science

DOI

UT WoS

000393601200010

Keywords in English

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

Tags

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

Abstract

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.

Links

EE2.3.30.0037, research and development project
Name: Zaměstnáním nejlepších mladých vědců k rozvoji mezinárodní spolupráce
GAP207/12/0775, research and development project
Name: Strukturně-funkční vztahy haloalkan dehalogenas
Investor: Czech Science Foundation
LH14027, research and development project
Name: Nové koncepty a nástroje pro racionální design enzymů
Investor: Ministry of Education, Youth and Sports of the CR
LO1214, research and development project
Name: Centrum pro výzkum toxických látek v prostředí (Acronym: RECETOX)
Investor: Ministry of Education, Youth and Sports of the CR
MUNI/M/1888/2014, interní kód MU
Name: Pokročilé hybridní metody studia transportních procesů v proteinech a jejich využití v designu biokatalyzátorů
Investor: Masaryk University, INTERDISCIPLINARY - Interdisciplinary research projects