SV AUC analysis for the GluA2 and GluA3 ATDs performed with different optical systems. Shown as pairs are the normalized sedimentation coefficient c(s) distributions (A, C, and E) and the sw isotherm (B, D, and F) derived by integration. All SV data and isotherm models are shown in units of experimental s-values. (A) c(s) distributions for EndoH-digested GluA2S measured at 230 nm. (B) Comparison of sw isotherms for the same data (black) and for GluA2S with complex glycosylation (red) acquired by absorbance at 230 nm (circles) and interference detection (diamonds). Fits for a monomer–dimer association were calculated with hydrodynamic constraints for monomer s-values of 2.75–3.37 S; the best-fit dimer s-values were 5.31 S (EndoH) and 5.07 S (293T), with Kd values of 5.6 and 8.3 nM, respectively. (C) Fluorescence-detected c(s) distributions for EndoH-digested FAM-labeled GluA2S (solid lines); the dotted line shows the c(s) distribution for absorbance detection at 495 nm of EndoH-digested FAM-labeled GluA2S. (D) sw isotherms for EndoH-digested GluA2S derived from integration of fluorescence-detected c(s) profiles for a dilution series (green) and a titration series with unlabeled protein (blue), with the global best-fit isotherm in the absence of hydrodynamic constraints (blue-green line). For comparison, sw isotherms were measured by absorbance at 230 and 280 nm for the same preparation before (red circles) and after FAM labeling (red triangle), respectively, and by absorbance at 495 nm from a different FAM-labeled preparation (black diamond). The best-fit s-value of the dimer was 5.44 S, but the monomer s-value was undefined, with a range from 2.75 to 3.37 S yielding statistically indistinguishable fits, indicated by the red lines for the extreme values, with the shaded area highlighting the range. (E and F) GluA3 c(s) distributions and the isotherm of sw values fit with a monomer–dimer Kd of 5.6 µM.
