Characterization of the structure of alpha-crystallin with the synthetic cross-linker 3,3\u27-dithiobis(sulfosuccinimidyl propionate)

Despite numerous efforts, the three-dimensional structure of the lens protein α-crystallin remains elusive. In this study, the structure of α-crystallin was investigated using the cross-linker 3,3-dithiobis(sulfosuccinimidyl propionate), DTSSP. Cross-linking reagents can react with side chains of amino acids that are within close proximity. Digestion of the cross-linked proteins and mass spectrometric analysis of the resulting peptides can identify the cross-linked residues, thus providing insight into the folded structures of proteins. However, our initial analysis of α-crystallin cross-linked with DTSSP was difficult because many ions in the mass spectra could not be attributed to the expected reactions of DTSSP. To better understand its reactivity, products from the reaction of DTSSP with several model peptides were analyzed by HPLC electrospray ionization mass spectrometry. Several products not previously reported were identified. Sources for these unexpected products were traced to reaction of DTSSP with ammonium ions in the buffer, to reaction of contaminants present in the commercial DTSSP reagent, and to unexpected reactivity of DTSSP with serine and tyrosine residues. In addition, the collision-induced-dissociation of peptides modified by DTSSP showed that certain DTSSP-peptide adducts easily undergo in-source fragmentation to give additional, unexpected ions. This study of the reactions of DTSSP with model peptides revealed the major types of ions that are likely to be found in proteolytic digests of proteins cross-linked with DTSSP, therefore facilitating the identification of residues in α-crystallin that were modified by DTSSP. Examination of the modifications of αA and αB treated with DTSSP showed multiple modifications of several lysines in both subunits, suggesting that some regions of αA and βB were readily accessed by DTSSP. However, the lack of modification at other lysines in the hydrophobic regions of αA and βB suggests that the accessibility of the cross-linker was limited. In addition, an intermolecular cross-link was detected between the C-termini of αA and βB. Evidence that these regions are within 12 Å in the folded protein supports some of the structural models proposed for α-crystallin