Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

Elizabeth M Boehm; Shyamal Subramanyam; Mohamed Ghoneim; M. Todd Washington; Maria Spies

doi:10.1016/bs.mie.2016.08.019

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Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

Journal article

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Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy

Elizabeth M Boehm, Shyamal Subramanyam, Mohamed Ghoneim, M. Todd Washington and Maria Spies

Methods in enzymology, Vol.581, pp.105-145

2016

DOI: 10.1016/bs.mie.2016.08.019

PMCID: PMC5403009

PMID: 27793278

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https://www.ncbi.nlm.nih.gov/pmc/articles/5403009View

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Abstract

Large, dynamic macromolecular complexes play essential roles in many cellular processes. Knowing how the components of these complexes associate with one another and undergo structural rearrangements is critical to understanding how they function. Single-molecule total internal reflection fluorescence (TIRF) microscopy is a powerful approach for addressing these fundamental issues. In this article, we first discuss single-molecule TIRF microscopes and strategies to immobilize and fluorescently label macromolecules. We then review the use of single-molecule TIRF microscopy to study the formation of binary macromolecular complexes using one-color imaging and inhibitors. We conclude with a discussion of the use of TIRF microscopy to examine the formation of higher-order ( i.e. , ternary, quaternary, etc.) complexes using multi-color setups. The focus throughout this article is on experimental design, controls, data acquisition, and data analysis. We hope that single-molecule TIRF microscopy, which has largely been the province of specialists, will soon become as common in the tool box of biophysicists and biochemists as structural approaches has become today.

co-localization single-molecule imaging

protein-protein and protein-nucleic acid interaction

Total Internal Reflection Fluorescence Microscopy (TIRFM)

single-molecule fluorescence

competitive and non-competitive inhibition

kinetic event resolving algorithm (KERA)

Details

Title: Subtitle: Quantifying the assembly of multicomponent molecular machines by single-molecule total internal reflection fluorescence microscopy
Creators: Elizabeth M Boehm - Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Shyamal Subramanyam - Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Mohamed Ghoneim - Biophysics Department, Faculty of Science, Cairo University, Egypt
M. Todd Washington - Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Maria Spies - Department of Biochemistry, University of Iowa Carver College of Medicine, Iowa City, IA, USA
Resource Type: Journal article
Publication Details: Methods in enzymology, Vol.581, pp.105-145
DOI: 10.1016/bs.mie.2016.08.019
PMID: 27793278
PMCID: PMC5403009
NLM abbreviation: Methods Enzymol
ISSN: 0076-6879
eISSN: 1557-7988
Language: English
Date published: 2016
Academic Unit: Radiation Oncology; Anesthesia; Biochemistry and Molecular Biology
Record Identifier: 9984024528702771

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