FIGURE 5.

SDS-PAGE and data analysis show that Cp183-WT capsids are digested faster than Cp183-EEE capsids and identify two populations of CTDs. A, Tris-Tricine SDS-PAGE of trypsin-cleaved Cp183-WT and Cp183-EEE T=4 capsids, stained with SYPRO® Ruby fluorescent protein stain. Trypsin digestion of Cp183 capsids was initiated, and aliquots were taken after 1–120 min and rapidly heated to 80 °C to stop digestion. An undigested capsid control (indicated by c) was loaded as a standard. Numbers on the right indicate putative cleavage products based on LC-MS results. B, SDS-PAGE results are plotted as percentage of Cp183-monomer versus time. The Cp183-WT data were fit to a function containing two first order decays: Equation 2 with a root mean square difference of 3.5%, showing that 25 and 75% of Cp183-WT CTDs were digested with a half-life of 28.8 min 2.5 min, respectively. The Cp183-EEE data were also fit to a function containing two first order decays: Equation 3 with a root mean square difference of 4.4%, showing that 50% of EEE C termini are digested with a half-life of 89.6 and 1.5 min, respectively. Each data point represents the average of two independent experiments, and the error bars correspond to the range observed.