skip to main content
DOE PAGES title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase

Abstract

Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here in this paper, we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 $μ$M range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s -1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidatemore » the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.« less

Authors:
 [1];  [2];  [2]; ORCiD logo [1]
  1. Stanford Univ., CA (United States). Dept. of Chemistry
  2. Arizona State Univ., Tempe, AZ (United States)
Publication Date:
Research Org.:
Stanford Univ., CA (United States); Arizona State Univ., Tempe, AZ (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1512950
Alternate Identifier(s):
OSTI ID: 1416215
Grant/Contract Number:  
FG02-07ER15892; FG02-09ER16123
Resource Type:
Accepted Manuscript
Journal Name:
Journal of Chemical Physics
Additional Journal Information:
Journal Volume: 148; Journal Issue: 12; Journal ID: ISSN 0021-9606
Publisher:
American Institute of Physics (AIP)
Country of Publication:
United States
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Wang, Quan, Serban, Andrew J., Wachter, Rebekka M., and Moerner, W. E. Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase. United States: N. p., 2018. Web. doi:10.1063/1.5005930.
Wang, Quan, Serban, Andrew J., Wachter, Rebekka M., & Moerner, W. E. Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase. United States. doi:10.1063/1.5005930.
Wang, Quan, Serban, Andrew J., Wachter, Rebekka M., and Moerner, W. E. Wed . "Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase". United States. doi:10.1063/1.5005930. https://www.osti.gov/servlets/purl/1512950.
@article{osti_1512950,
title = {Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase},
author = {Wang, Quan and Serban, Andrew J. and Wachter, Rebekka M. and Moerner, W. E.},
abstractNote = {Oligomerization plays an important role in the function of many proteins, but a quantitative picture of the oligomer distribution has been difficult to obtain using existing techniques. Here in this paper, we describe a method that combines sub-stoichiometric labeling and recently developed single-molecule diffusometry to measure the size distribution of oligomers under equilibrium conditions in solution, one molecule at a time. We use this technique to characterize the oligomerization behavior of Nicotiana tabacum (Nt) Rubisco activase (Nt-Rca), a chaperone-like AAA-plus ATPase essential in regulating carbon fixation during photosynthesis. We directly observed monomers, dimers, and a tetramer/hexamer mixture and extracted their fractional abundance as a function of protein concentration. We show that the oligomerization pathway of Nt-Rca is nucleotide dependent: ATPγS binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10 $μ$M range. Furthermore, we directly observed dynamic assembly and disassembly processes of single complexes in real time and from there estimated the rate of subunit exchange to be ∼0.1 s-1 with ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by >2 fold. These observations provide a quantitative starting point to elucidate the structure-function relations of Nt-Rca complexes. We envision the method to fill a critical gap in defining and quantifying protein assembly pathways in the small-oligomer regime.},
doi = {10.1063/1.5005930},
journal = {Journal of Chemical Physics},
number = 12,
volume = 148,
place = {United States},
year = {2018},
month = {3}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record

Citation Metrics:
Cited by: 5 works
Citation information provided by
Web of Science

Figures / Tables:

FIG. 1 FIG. 1: Measurement scheme. (a) Workflow of the assay based on single-molecule diffusometry. See the main text for details. (b) Structural model of the closed-ring hexamer of truncated Nt-Rca (PDB: 3ZW6). Full-length protein chains were measured in this work.

Save / Share:

Works referenced in this record:

Protein aggregation and neurodegenerative disease
journal, July 2004

  • Ross, Christopher A.; Poirier, Michelle A.
  • Nature Medicine, Vol. 10, Issue S7
  • DOI: 10.1038/nm1066

Regulation of Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase: PRODUCT INHIBITION, COOPERATIVITY, AND MAGNESIUM ACTIVATION
journal, August 2015

  • Hazra, Suratna; Henderson, J. Nathan; Liles, Kevin
  • Journal of Biological Chemistry, Vol. 290, Issue 40
  • DOI: 10.1074/jbc.m115.651745

Nucleotide Binding and Conformational Switching in the Hexameric Ring of a AAA+ Machine
journal, April 2013


Protein Oligomerization Monitored by Fluorescence Fluctuation Spectroscopy: Self-Assembly of Rubisco Activase
journal, September 2012

  • Chakraborty, Manas; Kuriata, Agnieszka M.; Nathan Henderson, J.
  • Biophysical Journal, Vol. 103, Issue 5
  • DOI: 10.1016/j.bpj.2012.07.034

Single-Molecule characterization of oligomerization kinetics and equilibria of the tumor suppressor p53
journal, November 2010

  • Rajagopalan, S.; Huang, F.; Fersht, A. R.
  • Nucleic Acids Research, Vol. 39, Issue 6
  • DOI: 10.1093/nar/gkq800

Dynamic light scattering: a practical guide and applications in biomedical sciences
journal, October 2016


Breaking the concentration limit of optical single-molecule detection
journal, January 2014

  • Holzmeister, Phil; Acuna, Guillermo P.; Grohmann, Dina
  • Chem. Soc. Rev., Vol. 43, Issue 4
  • DOI: 10.1039/c3cs60207a

Sensing cooperativity in ATP hydrolysis for single multisubunit enzymes in solution
journal, September 2011

  • Jiang, Y.; Douglas, N. R.; Conley, N. R.
  • Proceedings of the National Academy of Sciences, Vol. 108, Issue 41
  • DOI: 10.1073/pnas.1112244108

Small-angle scattering studies of biological macromolecules in solution
journal, September 2003


Elucidating the Aggregation Number of Dopamine-Induced α-Synuclein Oligomeric Assemblies
journal, January 2014

  • Zijlstra, Niels; Claessens, Mireille M. A. E.; Blum, Christian
  • Biophysical Journal, Vol. 106, Issue 2
  • DOI: 10.1016/j.bpj.2013.12.009

Assembly Pathway of an AAA+ Protein:  Tracking ClpA and ClpAP Complex Formation in Real Time
journal, May 2007

  • Kress, Wolfgang; Mutschler, Hannes; Weber-Ban, Eilika
  • Biochemistry, Vol. 46, Issue 21
  • DOI: 10.1021/bi602616t

Design principles of biochemical oscillators
journal, October 2008

  • Novák, Béla; Tyson, John J.
  • Nature Reviews Molecular Cell Biology, Vol. 9, Issue 12
  • DOI: 10.1038/nrm2530

AAA+ proteins: have engine, will work
journal, July 2005

  • Hanson, Phyllis I.; Whiteheart, Sidney W.
  • Nature Reviews Molecular Cell Biology, Vol. 6, Issue 7
  • DOI: 10.1038/nrm1684

Rubisco activity and regulation as targets for crop improvement
journal, November 2012

  • Parry, M. A. J.; Andralojc, P. J.; Scales, J. C.
  • Journal of Experimental Botany, Vol. 64, Issue 3
  • DOI: 10.1093/jxb/ers336

Structure of green-type Rubisco activase from tobacco
journal, November 2011

  • Stotz, Mathias; Mueller-Cajar, Oliver; Ciniawsky, Susanne
  • Nature Structural & Molecular Biology, Vol. 18, Issue 12
  • DOI: 10.1038/nsmb.2171

Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites
journal, August 1997


Biophysical characterization of higher plant Rubisco activase
journal, January 2013

  • Henderson, J. Nathan; Hazra, Suratna; Dunkle, Alison M.
  • Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, Vol. 1834, Issue 1
  • DOI: 10.1016/j.bbapap.2012.09.006

Nucleotide Dependence of Subunit Rearrangements in Short-Form Rubisco Activase from Spinach
journal, August 2017


An Oxygen Scavenging System for Improvement of Dye Stability in Single-Molecule Fluorescence Experiments
journal, March 2008

  • Aitken, Colin Echeverría; Marshall, R. Andrew; Puglisi, Joseph D.
  • Biophysical Journal, Vol. 94, Issue 5
  • DOI: 10.1529/biophysj.107.117689

Detection of Intensity Change Points in Time-Resolved Single-Molecule Measurements
journal, January 2005

  • Watkins, Lucas P.; Yang, Haw
  • The Journal of Physical Chemistry B, Vol. 109, Issue 1
  • DOI: 10.1021/jp0467548

Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson’s disease genetically related mutants
journal, November 2015

  • Tosatto, Laura; Horrocks, Mathew H.; Dear, Alexander J.
  • Scientific Reports, Vol. 5, Issue 1
  • DOI: 10.1038/srep16696

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations
journal, November 2014

  • Kuriata, Agnieszka M.; Chakraborty, Manas; Henderson, J. Nathan
  • Biochemistry, Vol. 53, Issue 46
  • DOI: 10.1021/bi500968h

Structural Symmetry and Protein Function
journal, June 2000


Small Oligomers of Ribulose-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Are Required for Biological Activity
journal, May 2013

  • Keown, Jeremy R.; Griffin, Michael D. W.; Mertens, Haydyn D. T.
  • Journal of Biological Chemistry, Vol. 288, Issue 28
  • DOI: 10.1074/jbc.m113.466383

An efficient system for high-level expression and easy purification of authentic recombinant proteins
journal, May 2004

  • Catanzariti, Ann-Maree; Soboleva, Tatiana A.; Jans, David A.
  • Protein Science, Vol. 13, Issue 5
  • DOI: 10.1110/ps.04618904

Native Mass Spectrometry: What is in the Name?
journal, December 2016

  • Leney, Aneika C.; Heck, Albert J. R.
  • Journal of The American Society for Mass Spectrometry, Vol. 28, Issue 1
  • DOI: 10.1007/s13361-016-1545-3

Direct characterization of amyloidogenic oligomers by single-molecule fluorescence
journal, September 2008

  • Orte, A.; Birkett, N. R.; Clarke, R. W.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 38
  • DOI: 10.1073/pnas.0803086105

Prediction of diffusion coefficients of proteins
journal, February 1990

  • Tyn, Myo T.; Gusek, Todd W.
  • Biotechnology and Bioengineering, Vol. 35, Issue 4
  • DOI: 10.1002/bit.260350402

Structure of Arabidopsis thaliana Rubisco activase
journal, March 2015

  • Hasse, Dirk; Larsson, Anna M.; Andersson, Inger
  • Acta Crystallographica Section D Biological Crystallography, Vol. 71, Issue 4
  • DOI: 10.1107/s1399004715001182

Single-molecule motions enable direct visualization of biomolecular interactions in solution
journal, March 2014


Rubisco Activases: AAA+ Chaperones Adapted to Enzyme Repair
journal, April 2017

  • Bhat, Javaid Y.; Thieulin-Pardo, Gabriel; Hartl, F. Ulrich
  • Frontiers in Molecular Biosciences, Vol. 4
  • DOI: 10.3389/fmolb.2017.00020

Prediction of Hydrodynamic and Other Solution Properties of Rigid Proteins from Atomic- and Residue-Level Models
journal, August 2011


Resolution of Fluorescence Correlation Measurements
journal, March 1999


Mg2+ and ATP or adenosine 5′-[γ-thio]-triphosphate (ATPγS) enhances intrinsic fluorescence and induces aggregation which increases the activity of spinach Rubisco activase
journal, September 1993

  • Wang, Zhen Y.; Ramage, Robert T.; Portis, Archie R.
  • Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1202, Issue 1
  • DOI: 10.1016/0167-4838(93)90061-u

Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid β-peptide
journal, February 2007

  • Haass, Christian; Selkoe, Dennis J.
  • Nature Reviews Molecular Cell Biology, Vol. 8, Issue 2
  • DOI: 10.1038/nrm2101

    Figures/Tables have been extracted from DOE-funded journal article accepted manuscripts.