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

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

doi:10.1063/1.5005930

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

Journal Article · Wed Mar 28 00:00:00 UTC 2018 · Journal of Chemical Physics

DOI: https://doi.org/10.1063/1.5005930 · OSTI ID:1512950

Wang, Quan ^[1]; Serban, Andrew J. ^[2]; Wachter, Rebekka M. ^[2];

^[3]

Stanford Univ., CA (United States). Dept. of Chemistry; none
Arizona State Univ., Tempe, AZ (United States)
Stanford Univ., CA (United States). Dept. of Chemistry

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.

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Research Organization:: Arizona State Univ., Tempe, AZ (United States); Stanford Univ., CA (United States)

Sponsoring Organization:: USDOE; USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)

Grant/Contract Number:: FG02-07ER15892; SC0002423

OSTI ID:: 1512950

Journal Information:: Journal of Chemical Physics, Journal Name: Journal of Chemical Physics Journal Issue: 12 Vol. 148; ISSN 0021-9606

Publisher:: American Institute of Physics (AIP)Copyright Statement

Country of Publication:: United States

Language:: English

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

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