Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps

Benjamin Vermeer; Jannick van Ossenbruggen; Sonja Schmid

doi:10.1007/978-1-0716-3377-9_13

Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps

Benjamin Vermeer
, Jannick van Ossenbruggen
, Sonja Schmid^*

^*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceeding › Chapter › Academic › peer-review

2 Citations (Scopus)

Abstract

Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system – notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.

Original language	English
Title of host publication	Methods in Molecular Biology
Publisher	Humana Press
Pages	267-291
Number of pages	25
Volume	2694
ISBN (Electronic)	9781071633779
ISBN (Print)	9781071633762
DOIs	https://doi.org/10.1007/978-1-0716-3377-9_13
Publication status	Published - 2024

Publication series

Name	Methods in Molecular Biology
Volume	2694
ISSN (Print)	1064-3745
ISSN (Electronic)	1940-6029

Keywords

Bioconjugation
Chaperone protein Hsp90
Protein dynamics
Protein purification
Single-molecule FRET
Surface functionalization

Access to Document

10.1007/978-1-0716-3377-9_13

https://edepot.wur.nl/641764Licence: Taverne

Single-Molecule FRET-Resolved Protein Dynamics - from Plasmid to Data in Six Steps
Vermeer, B. (Creator), van Ossenbruggen, J. (Creator) & Schmid, S. (Creator), Wageningen University & Research, 3 Jul 2023
DOI: 10.5281/zenodo.7273265
Dataset

Cite this

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title = "Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps",

abstract = "Single-molecule F{\"o}rster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system – notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.",

keywords = "Bioconjugation, Chaperone protein Hsp90, Protein dynamics, Protein purification, Single-molecule FRET, Surface functionalization",

author = "Benjamin Vermeer and \{van Ossenbruggen\}, Jannick and Sonja Schmid",

year = "2024",

doi = "10.1007/978-1-0716-3377-9\_13",

language = "English",

isbn = "9781071633762",

volume = "2694",

series = "Methods in Molecular Biology",

publisher = "Humana Press",

pages = "267--291",

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T1 - Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps

AU - Vermeer, Benjamin

AU - van Ossenbruggen, Jannick

AU - Schmid, Sonja

PY - 2024

Y1 - 2024

N2 - Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system – notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.

AB - Single-molecule Förster resonance energy transfer (smFRET) is a powerful technique for the detection of conformational dynamics of biomolecules. While many smFRET experiments are performed using dye-labeled DNA, here we describe a comprehensive protocol to resolve the conformational dynamics of a protein system – notably from plasmid to data. Using the example of the heat-shock protein Hsp90, we describe the protein production and threefold site-specific bioconjugation, the smFRET measurement using total internal reflection fluorescence microscopy (TIRFM), and raw data processing to reveal time-resolved protein dynamics. The described smFRET approach is readily transferrable to the study of many more all-protein systems and their conformational energy landscape.

KW - Bioconjugation

KW - Chaperone protein Hsp90

KW - Protein dynamics

KW - Protein purification

KW - Single-molecule FRET

KW - Surface functionalization

U2 - 10.1007/978-1-0716-3377-9_13

DO - 10.1007/978-1-0716-3377-9_13

M3 - Chapter

C2 - 37824009

AN - SCOPUS:85175149712

SN - 9781071633762

VL - 2694

T3 - Methods in Molecular Biology

SP - 267

EP - 291

BT - Methods in Molecular Biology

PB - Humana Press

ER -

Single-Molecule FRET-Resolved Protein Dynamics – from Plasmid to Data in Six Steps

Abstract

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Single-Molecule FRET-Resolved Protein Dynamics - from Plasmid to Data in Six Steps

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