Cross-Linking-Based Flexibility and Proximity Relationships between the TM Segments of the Escherichia coli YidC

The YidC family members function to insert proteins into membranes in bacteria, chloroplasts, and mitochondria, and they can also act as a platform to fold and assemble proteins into higher-order complexes. Here, we provide information about the proximity relationships and dynamics of the five conserved C-terminal transmembrane (TM) regions within Escherichia coli YidC. By using a YidC construct with tandem thrombin protease sites introduced into the cytoplasmic loop C1, cross-linking between paired-Cys residues located within TM segments or in the membrane border regions was studied using thio-specific homobifunctional cross-linking agents with different spanner lengths or by iodine-catalyzed disulfide formation. These in vivo cross-linking studies that can detect transient interactions and different conformational states of the protein show that TM3, TM4, TM5, and TM6 each have a face oriented toward TM2 of the in vivo expressed YidC. The studies also reveal that YidC is a dynamic protein, as cross-link...