FIGURE 4.
Cross-linking to position 114 in the AST domain of GspB736FLAG. GspB736FLAG/L114TAG was expressed from pCDF, the BzF-specific amber suppression components (amino-acyl tRNA synthase and tRNACUA) were expressed from pEVOL-pBpF, and the aSec operon was expressed from pCOLAaSec/gtfA::IS10. GspB736FLAG was detected by Western blotting with anti-FLAG antibodies, and the full-length preprotein is indicated by a black arrow. Red asterisks indicate cross-linked complexes that occur in both the presence and absence of the aSec system. Red arrow indicates an aSec-specific cross-linked complex. The panel on the right shows a shorter exposure of the Western blot, in which the 105 kDa cross-linked complex is more readily visible.