PT - JOURNAL ARTICLE AU - Quan Wang AU - Andrew J. Serban AU - Rebekka M. Wachter AU - W.E. Moerner TI - Single-molecule diffusometry reveals the nucleotide-dependent oligomerization pathways of <em>Nicotiana tabacum</em> Rubisco activase AID - 10.1101/191742 DP - 2017 Jan 01 TA - bioRxiv PG - 191742 4099 - http://biorxiv.org/content/early/2017/09/20/191742.short 4100 - http://biorxiv.org/content/early/2017/09/20/191742.full AB - 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 1-10μM 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 ATPγS. On the other hand, ADP binding destabilizes Rca complexes by enhancing the rate of subunit exchange by &gt;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.