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Title: Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding

Abstract

While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates.

Authors:
 [1];  [2]
  1. DePauw Univ., Greencastle, IN (United States)
  2. Univ. of California, Berkeley, CA (United States); Chan Zuckerburg Biohub, San Francisco, CA (United States)
Publication Date:
Research Org.:
Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1571996
Grant/Contract Number:  
AC02-05CH11231
Resource Type:
Accepted Manuscript
Journal Name:
Methods and Protocols
Additional Journal Information:
Journal Volume: 2; Journal Issue: 2; Journal ID: ISSN 2409-9279
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; force spectroscopy; optical tweezers; chemo-mechanical unfolding; protein folding; denaturant; urea

Citation Formats

Guinn, Emily J., and Marqusee, Susan. Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding. United States: N. p., 2019. Web. doi:10.3390/mps2020032.
Guinn, Emily J., & Marqusee, Susan. Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding. United States. doi:10.3390/mps2020032.
Guinn, Emily J., and Marqusee, Susan. Sat . "Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding". United States. doi:10.3390/mps2020032. https://www.osti.gov/servlets/purl/1571996.
@article{osti_1571996,
title = {Using Single-Molecule Chemo-Mechanical Unfolding to Simultaneously Probe Multiple Structural Parameters in Protein Folding},
author = {Guinn, Emily J. and Marqusee, Susan},
abstractNote = {While single-molecule force spectroscopy has greatly advanced the study of protein folding, there are limitations to what can be learned from studying the effect of force alone. We developed a novel technique, chemo-mechanical unfolding, that combines multiple perturbants—force and chemical denaturant—to more fully characterize the folding process by simultaneously probing multiple structural parameters—the change in end-to-end distance, and solvent accessible surface area. Here, we describe the theoretical background, experimental design, and data analysis for chemo-mechanical unfolding experiments probing protein folding thermodynamics and kinetics. This technique has been applied to characterize parallel protein folding pathways, the protein denatured state, protein folding on the ribosome, and protein folding intermediates.},
doi = {10.3390/mps2020032},
journal = {Methods and Protocols},
number = 2,
volume = 2,
place = {United States},
year = {2019},
month = {4}
}

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Works referenced in this record:

Analysis of Single-Molecule FRET Trajectories Using Hidden Markov Modeling
journal, September 2006


Decoding the mechanical fingerprints of biomolecules
journal, October 2015


The Protein-Folding Problem, 50 Years On
journal, November 2012


ClpX(P) Generates Mechanical Force to Unfold and Translocate Its Protein Substrates
journal, April 2011


Mechanical Processes in Biochemistry
journal, June 2004


Single-Molecule Chemo-Mechanical Spectroscopy Provides Structural Identity of Folding Intermediates
journal, March 2016

  • Motlagh, Hesam N.; Toptygin, Dmitri; Kaiser, Christian M.
  • Biophysical Journal, Vol. 110, Issue 6
  • DOI: 10.1016/j.bpj.2015.12.042

Direct observation of multiple misfolding pathways in a single prion protein molecule
journal, March 2012

  • Yu, H.; Liu, X.; Neupane, K.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 14
  • DOI: 10.1073/pnas.1107736109

Force-dependent switch in protein unfolding pathways and transition-state movements
journal, January 2016

  • Zhuravlev, Pavel I.; Hinczewski, Michael; Chakrabarti, Shaon
  • Proceedings of the National Academy of Sciences, Vol. 113, Issue 6
  • DOI: 10.1073/pnas.1515730113

Exploring the Denatured State Ensemble by Single-Molecule Chemo-Mechanical Unfolding: The Effect of Force, Temperature, and Urea
journal, February 2018


Probing the protein-folding mechanism using denaturant and temperature effects on rate constants
journal, September 2013

  • Guinn, E. J.; Kontur, W. S.; Tsodikov, O. V.
  • Proceedings of the National Academy of Sciences, Vol. 110, Issue 42
  • DOI: 10.1073/pnas.1311948110

The Ribosome Modulates Nascent Protein Folding
journal, December 2011


Pause sequences facilitate entry into long-lived paused states by reducing RNA polymerase transcription rates
journal, July 2018


Protein folding and unfolding under force: Protein Folding and Unfolding Under Force
journal, August 2013

  • Jagannathan, Bharat; Marqusee, Susan
  • Biopolymers, Vol. 99, Issue 11
  • DOI: 10.1002/bip.22321

A small single-domain protein folds through the same pathway on and off the ribosome
journal, November 2018

  • Guinn, Emily J.; Tian, Pengfei; Shin, Mia
  • Proceedings of the National Academy of Sciences, Vol. 115, Issue 48
  • DOI: 10.1073/pnas.1810517115

Determination of thermodynamics and kinetics of RNA reactions by force
journal, October 2006

  • Tinoco, Ignacio; T. X. Li, Pan; Bustamante, Carlos
  • Quarterly Reviews of Biophysics, Vol. 39, Issue 4
  • DOI: 10.1017/S0033583506004446

Theory, analysis, and interpretation of single-molecule force spectroscopy experiments
journal, October 2008

  • Dudko, O. K.; Hummer, G.; Szabo, A.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 41
  • DOI: 10.1073/pnas.0806085105

Mechanisms of Cellular Proteostasis: Insights from Single-Molecule Approaches
journal, May 2014


Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy
journal, May 2014


The folding of an enzyme
journal, April 1992


Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo
journal, April 2015


The folding of single domain proteins—have we reached a consensus?
journal, February 2011


Quantifying why urea is a protein denaturant, whereas glycine betaine is a protein stabilizer
journal, September 2011

  • Guinn, E. J.; Pegram, L. M.; Capp, M. W.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 41
  • DOI: 10.1073/pnas.1109372108

Mechanical Biochemistry of Proteins One Molecule at a Time
journal, January 2008

  • Oberhauser, Andres F.; Carrión-Vázquez, Mariano
  • Journal of Biological Chemistry, Vol. 283, Issue 11
  • DOI: 10.1074/jbc.R700050200

Models for the specific adhesion of cells to cells
journal, May 1978


The Complex Folding Network of Single Calmodulin Molecules
journal, October 2011


Evolution, energy landscapes and the paradoxes of protein folding
journal, December 2015


Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein
journal, September 2012

  • Jagannathan, B.; Elms, P. J.; Bustamante, C.
  • Proceedings of the National Academy of Sciences, Vol. 109, Issue 44
  • DOI: 10.1073/pnas.1201800109

The folding cooperativity of a protein is controlled by its chain topology
journal, May 2010

  • Shank, Elizabeth A.; Cecconi, Ciro; Dill, Jesse W.
  • Nature, Vol. 465, Issue 7298
  • DOI: 10.1038/nature09021

Distinguishing Signatures of Multipathway Conformational Transitions
journal, February 2017


The ribosome destabilizes native and non-native structures in a nascent multidomain protein: EF-G Folding on the Ribosome
journal, May 2017

  • Liu, Kaixian; Rehfus, Joseph E.; Mattson, Elliot
  • Protein Science, Vol. 26, Issue 7
  • DOI: 10.1002/pro.3189

Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein
journal, April 2015

  • Guinn, Emily J.; Jagannathan, Bharat; Marqusee, Susan
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms7861

Denaturant m values and heat capacity changes: Relation to changes in accessible surface areas of protein unfolding
journal, October 1995

  • Myers, Jeffrey K.; Nick Pace, C.; Martin Scholtz, J.
  • Protein Science, Vol. 4, Issue 10
  • DOI: 10.1002/pro.5560041020

Theory of protein folding
journal, February 2004

  • Onuchic, José Nelson; Wolynes, Peter G.
  • Current Opinion in Structural Biology, Vol. 14, Issue 1
  • DOI: 10.1016/j.sbi.2004.01.009

A simple DNA handle attachment method for single molecule mechanical manipulation experiments: Biomechanics of Biological Systems
journal, June 2016

  • Min, Duyoung; Arbing, Mark A.; Jefferson, Robert E.
  • Protein Science, Vol. 25, Issue 8
  • DOI: 10.1002/pro.2952

A Highly Compliant Protein Native State with a Spontaneous-like Mechanical Unfolding Pathway
journal, October 2012

  • Heidarsson, Pétur O.; Valpapuram, Immanuel; Camilloni, Carlo
  • Journal of the American Chemical Society, Vol. 134, Issue 41
  • DOI: 10.1021/ja305862m