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 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.
- Authors:
-
[1]
- Stanford Univ., CA (United States). Dept. of Chemistry
- 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)
- 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. https://doi.org/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. https://doi.org/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 = {Wed Mar 28 00:00:00 EDT 2018},
month = {Wed Mar 28 00:00:00 EDT 2018}
}
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Figures / Tables:
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.
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