ECAM in native, methylamine-treated, and protease-activated forms by small angle X-ray scattering at physiological pH. ECAM changes conformation upon activation SAXS experiments were performed with four distinct samples: native ECAM, as well as ECAM reacted with methylamine, chymotrypsin, and elastase. All samples were purified by gel filtration chromatography. All activated forms of ECAM migrate faster than the native form in non-denaturing PAGE, suggesting that activation induces a conformational change and confirming the existence of electrophoretically `fast’ forms of bacterial a2Ms. Notably, the transition from `slow’ to `fast’ forms by eukaryotic a-macroglobulins results in a considerable modification of the overall structure of the dimeric and tetrameric molecules, revealing that the interplay between bait region and thioester cleavage plays key roles in the induction of conformational changes. Scattering patterns were recorded at different ECAM concentrations for all four samples and did not suggest any oligomerization or aggregation events, and are shown in Fig. 3B. The data are 212141-51-0 site represented with the form log I versus s, where I is the measured intensity and s is the scattering angle. The intensity curve for native ECAM shows a distinct side maximum that shifts to higher angles after the protein is reacted with methylamine and suggests that upon activation, ECAM undergoes a conformational change. A qualitatively similar change was also reported for the scattering curves of native and methylamine-treated human a2M, albeit on a different scale. In the case of the elastase or chymotrypsin-treated forms, the side maximum is shifted towards higher angles, indicating a compactation of the native structure, which is in agreement with the decrease of the maximum distance in the p plots from 19 to approximately 16 nm. While the Dmax of the native and methylamine-activated forms were similar, it follows from Fig. 3B that the main maximum in the p curve displays a shift from 5.80 for native ECAM to 5.70 nm for the methylamine-treated form. In addition, there were significantly more differences in the range from 10 to 15 nm in the case of the methylamine-activated form with respect to the native form, which suggests a domain rearrangement in line with the increase of the Rg between both forms. Interestingly, this change was more substantial after incubation with proteases, where the maxima were at 5.28 and 5.25 nm for the chymotrypsin and elastase complexes, respectively. Therefore, the modification in Dmax, the shape of the p curve and a modified Rg all point to the fact that ECAM undergoes a conformational modification after reaction with methylamine, and this change is even more pronounced upon its reaction with proteases. Surprisingly, by employing fluorescence spectroscopy, Doan and Gettins recently concluded that ECAM does not undergo major structural modifications upon treatment with methylamine. The reasons for this 
discrepancy are unclear, but the results presented here from both EM and SAXS studies clearly show that a conformational modification occurs upon activation. The slow decline of the p functions at large distances in all samples might suggest that parts of the structure can adopt a second, lowly populated conformation or structural flexibility; this effect is most pronounced for the native and methylamine activated forms. However, it should be noted that in the absence of high-resolution models of ECAM our SAXS data alon