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Tim50 contains a C-terminal presequence-binding domain. (A) Schematic representation of Tim50 and truncation constructs used in this study. PS, presequence; TM, transmembrane domain; NIF, NIF domain; PBD, presequence-binding domain. (B) Purified Tim50IMS was incubated with presequence probes under UV irradiation for 30 min, samples analyzed by SDS-PAGE, and proteins subjected to in-gel digest before LC MALDI MS/MS analysis. PA, photo-adduct. (C) Indicated Tim50 constructs were purified from E. coli and analyzed by SDS-PAGE and stained with colloidal Coomassie. Asterisk denotes proteolytic Tim50PBD fragment. (D) Purified Tim50 variants were subjected to presequence photo cross-linking for 30 min and analyzed by Western blotting using anti-Tim50 antibodies. PA, photo-adduct. (E) Chemical cross-linking of pCox4 and SynB2 to isolated Tim50 variants using 100 µM DFDNB for 30 min on ice. Samples were analyzed as in D. (F) Photo cross-linking of presequences to Tim50PBD analyzed as in B. PA, photo-adduct. (G) Fragment ion mass spectrum of peptide Tim50422-435 cross-linked to pL19B18-24. Photo-adduct of pL19B with Tim50PBD (F) was digested with trypsin and subjected to LC-MALDI-MS/MS analysis. The indicated series of y- and b-ions revealed Met427 as the cross-link site. Signals of m/z 837.53 and 903.49 are frequently observed after fragmentation of cross-linked methionine side chains. (H) Alignment of Tim50 using ClustalW 2.0.11. Shown is the PBD domain; black, identical residues in four species, similar residues in at least four or three species are colored in dark or light gray, respectively. Similarity rules according to Erdmann et al. (1991). S.c., Saccharomyces cerevisiae; C.g., Candida glabrata; S.p., Schizosaccharomyces pombe; Y.l., Yarrowia lipolytica; N.c., Neurospora crassa. (I) Chemical cross-linking of 1 µM Tim50IMS to 5 µM pCox4 or SynB2 in the presence of increasing salt concentrations using 100 µM DFDNB for 30 min on ice. Samples were analyzed as in D.