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  Label-free optical detection of single enzyme-reactant reactions and associated conformational changes

Kim, E., Baaske, M. D., Schuldes, I., Wilsch, P. S., & Vollmer, F. (2017). Label-free optical detection of single enzyme-reactant reactions and associated conformational changes. Science Advances, 3(3): e1603044. doi:10.1126/sciadv.1603044.

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 Creators:
Kim, Eugene1, Author           
Baaske, Martin D.1, Author           
Schuldes, Isabel1, Author           
Wilsch, Peter S.1, Author           
Vollmer, Frank1, Author           
Affiliations:
1Vollmer Research Group, Research Groups, Max Planck Institute for the Science of Light, Max Planck Society, ou_2364719              

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Free keywords: conformational change; optical microcavity; polymerase; protein-DNA interactions; single enzyme dynamics; single molecule reaction; whispering Gallery mode
 Abstract: Monitoring the kinetics and conformational dynamics of single enzymes is crucial to better understand their biological functions because these motions and structural dynamics are usually unsynchronized among the molecules. However, detecting the enzyme-reactant interactions and associated conformational changes of the enzyme on a single-molecule basis remains as a challenge to established optical techniques because of the commonly required labeling of the reactants or the enzyme itself. The labeling process is usually nontrivial, and the labels themselves might skew the physical properties of the enzyme. We demonstrate an optical, label-free method capable of observing enzymatic interactions and associated conformational changes on a single-molecule level. We monitor polymerase/DNA interactions via the strong near-field enhancement provided by plasmonic nanorods resonantly coupled to whispering gallery modes in microcavities. Specifically, we use two different recognition schemes: one in which the kinetics of polymerase/DNA interactions are probed in the vicinity of DNA-functionalized nanorods, and the other in which these interactions are probed via the magnitude of conformational changes in the polymerase molecules immobilized on nanorods. In both approaches, we find that low and high polymerase activities can be clearly discerned through their characteristic signal amplitude and signal length distributions. Furthermore, the thermodynamic study of the monitored interactions suggests the occurrence of DNA polymerization. This work constitutes a proof-of-concept study of enzymatic activities using plasmonically enhanced microcavities and establishes an alternative and label-free method capable of investigating structural changes in single molecules.

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Language(s): eng - English
 Dates: 2016-12-052017-03-092017-03-29
 Publication Status: Published online
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1126/sciadv.1603044
 Degree: -

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Title: Science Advances
  Other : Sci. Adv.
Source Genre: Journal
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Publ. Info: Washington : AAAS
Pages: - Volume / Issue: 3 (3) Sequence Number: e1603044 Start / End Page: - Identifier: ISSN: 2375-2548
CoNE: https://pure.mpg.de/cone/journals/resource/2375-2548