Fig. 1.

Mapping the cross-linked sites on actin. (A) Actin monomers (m) and ACD cross-linked dimers (d) were subjected to limited proteolysis by GluC, trypsin, and subtilisin. The expected molecular mass of proteolytic peptides and the soybean trypsin inhibitor (in blue) are indicated. (B) Summary of the results from (A) with an indication of cleavage sites, the location of the covalent cross-link bond (yellow bar), and the masses of proteolytic peptides. Red and blue rectangles represent two protomers in the ACD cross-linked actin dimer. (C) Ribbon representation of two crystal structures of the ACD cross-linked actin dimer (AD) in complex with GS1 and with both GS1 and DNaseI (GS1 and DNaseI are not shown) and the recent Holmes model of actin filament (20). Both dimers are oriented in a way that displays better the proximity of the cross-liked peptides. Subdomains 1 to 4 of each protomer are colored blue, yellow, red, and green, respectively. Dotted black line indicates disordered residues 40–49. The cross-linked peptides 48–61 and 257–284, mapped by limited proteolysis and mass spectrometry of the biotin-labeled cross-linked peptide, are shown in black. Taking peptide 48–61 as the only constraint for the localization of one of the cross-linked residues, K50 and E270 were identified as the only two residues within 15-Å radius from each other in both crystal structures as detailed in text. (D) Tryptic peptides of cross-linked actin dimer were analyzed by ESI-FTMS. The 1095.9 (M + 5H)⁵⁺ peak in MS spectra, corresponding to the cross-linked peptide, was fragmented in the FT-ICR cell. The most intense of the 104 ions that could be matched to the sequence are annotated in blue (listed in Tables S1 and S2). The masses from resulting high resolution MS/MS spectrum were assigned to peptide fragments by using MS2Assign software. The inset shows the base line separation of the quintuply charged remainder of the precursor. (E) Schematic representation of the cross-linked peptides with the number of ions found for each bond. Of 47 bonds, product ions defining 43 were found. Importantly, 10 ions can be found related to a fragmentation next to the amino acids involved in the cross-linking. This identifies unambiguously the site of cross-linking.