Cross-linking TM1 to TM2. (A) Schematic of engineered β1. Mutations G12C and R182C were made in a background of HIS-FLAG-pWT β1 with the additional mutation E13Q. In this construct, the first site susceptible to cleavage by GluC endoproteinase is C-terminal to TM1 at E50. Also, the native E143, just N-terminal to TM2, is the last E in the sequence. There are five native E’s between E50 and E143, but they do not affect the outcome; complete cleavage by GluC results in an N-terminal 7,747-D fragment containing TM1 and a C-terminal 5,406-D fragment containing TM2. Their cross-linking results in a 13,153-D fragment. (B) Immunoblot with anti-FLAG antibody of β1 G12C-R182C after cross-linking with 400 µM QPD in 0.01% saponin and cleavage with GluC (see Materials and methods). Capture and immunoblotting methods were similar to those published previously (Liu et al., 2010; Wu et al., 2013). Cleavage with GluC after cross-linking yielded both an ∼13-kD band and an ∼7.7-kD band. The extent of cross-linking is calculated as (density 13-kD band)/(density 13-kD band + density 7.7-kD band). After DTT reduction, only a 7.7-kD band is seen, showing that GluC cleavage was complete.