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

Quan Wang, Andrew J. Serban, Rebekka Wachter, W. E. Moerner

Research output: Contribution to journalArticle

6 Citations (Scopus)

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 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 μM 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.

Original languageEnglish (US)
Article number123319
JournalJournal of Chemical Physics
Volume148
Issue number12
DOIs
StatePublished - Mar 28 2018

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Ribulose-Bisphosphate Carboxylase
Oligomerization
nucleotides
Tissue Plasminogen Activator
oligomers
Oligomers
Nucleotides
proteins
Dimers
Molecules
assembly
dimers
molecules
adenosine diphosphate
Proteins
photosynthesis
Photosynthesis
Adenosine Diphosphate
Labeling
marking

ASJC Scopus subject areas

  • Physics and Astronomy(all)
  • Physical and Theoretical Chemistry

Cite this

Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase. / Wang, Quan; Serban, Andrew J.; Wachter, Rebekka; Moerner, W. E.

In: Journal of Chemical Physics, Vol. 148, No. 12, 123319, 28.03.2018.

Research output: Contribution to journalArticle

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