Fig. 2.
Nonmonotonic behavior in a cNup62 brush caused by Kapβ1-FG binding. (A) Successive 3·30 s BSA injections follow 16 cKapβ1 titrations ranging from 0.1 nM to 13.4 μM on a cNup62 brush characterized by gcNup62 = 2.4 nm and d2(initial) = 14.1 nm. (B) The cNup62 brush undergoes collapse at low ρKapβ1 (1), followed by recovery (2), which reaches pileup (3) upon crossing Δd equal 0. Included are the values of gKapβ1 and cKapβ1 (in parentheses) that correspond to each respective Δd measurement. (C) The steady-state (Req) SPR response across the entire cKapβ1 range (from A; 0.1 nM to 13.4 μM) is optimally fit using a two-component Langmuir isotherm (green) giving KD1 = 347 nM and KD2 = 95.9 μM. For single fits (KD of approximately 400 nM), χ2 is minimized at low terminal cKapβ1 values (grey, purple, and red) but deviates past cKapβ1 > 4 μM (blue and pink). Solid and dashed lines denote the actual fitted cKapβ1 range and the predicted KD behavior, respectively. (Inset) A single KD = 1.28 μM is found for Kapβ1 binding to sparse cNup62 mushrooms where gcNup62 = 11.0 nm and d2(initial) = 2.5 nm.