Figure 4.

Distinguishing between assembly states of nearly identical mass using CDMS. (A) The cpTMV-S65C mutant can both form disulfide bonds at the disk’s periphery and (B) assemble into stacks of four disks, resulting in two structures with a nearly equal mass at 1.2 MDa. The location of the S65C mutation is shown in red. (C) A mass histogram of cpTMV-S65C in 100 mM ammonium acetate solution oxidized in air displays assemblies with even-numbered disk stoichiometries varying from two to twelve disks per assembly. (D) After reduction with 1 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h, there is a decrease in the proportion of larger-sized assemblies of cpTMV-S65C. (E) A two-dimensional mass vs. charge histogram of air-oxidized cpTMV-S65C in 100 mM ammonium acetate solution displays two distinct populations at 1.2 MDa above and below the Rayleigh limit (red line). It also shows broad charge distributions for species with greater than four disks which indicate a mixture of different stacked and edge-on conformers. (F) After treatment with TCEP for 1 h, populations larger than four disks were no longer visible, and only a small population of the higher charged species at 1.2 MDa remained, indicating that this is the peripherally disulfide bonded species.