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bioRxiv · 10.1101/191742

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

Wang, Q.·Serban, A. J.·Wachter, R. M.·Moerner, W. E.
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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: ATP{gamma}S binding strongly promotes tetramer/hexamer formation from dimers and results in a preferred tetramer/hexamer population for concentrations in the 1-10M 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 ~0.1s-1 with ATP{gamma}S. 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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Wang, Q., Serban, A. J., Wachter, R. M., Moerner, W. E.. 2017-09-20. Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of Nicotiana tabacum Rubisco activase. https://doi.org/10.1101/191742

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