Chemistry of Periodate-Mediated Cross-Linking of 3,4-Dihydroxylphenylalanine-Containing Molecules to Proteins

Abstract

Chemical cross-linking is an attractive approach to map peptide-protein and protein-protein complexes. Previously, we explored 3,4-dihydroxylphenylalanine (DOPA) as a protein cross-linking agent upon periodate oxidation.(Burdine, L.; Gillette, T. G.; Lin, H-J.; Kodadek, T. J. Am. Chem. Soc. 2004, 126, 11442-11443) We report here a study on the chemistry of DOPA-protein crosslinking. First, using a peptide nucleic acid (PNA) templated system, we identified the α-amino, ε-amino of Lys, imidazole of His, and thiol of Cys as functional groups capable of attacking DOPA ortho-quinone. Second, we demonstrated that periodate-induced DOPA-protein cross-linking could be carried out efficiently at neutral pH in the presence of excess aliphatic 1,2-diols such as ethylene glycol, lactose, and ATP. This result indicated that DOPA-protein cross-linking and 1,2-diol oxidative cleavage proceed via different mechanisms and, that carbohydrates will not interfere with this process when carried out in crude cell extracts or on intact cells.Protein-protein interactions are of fundamental importance in almost all biological processes. In some cases, these are strong, stable contacts while in others they represent much weaker, transient interactions. In principle, chemical cross-linking followed by detailed product analysis is an attractive approach to map protein-protein contacts under native conditions. However, this is complicated by the low reactivity of typical bifunctional cross-linking reagents and their tendency to produce false positives (reviewed in references 1, 2 ). As part of a program to develop a new generation of more efficient and useful cross-linking reagents, we have shown recently that molecules containing 3,4-dihydroxylphenylalanine (DOPA) often cross-link efficiently to their protein receptors when treated with sodium periodate. 3 Under these conditions, DOPA is converted to an ortho-quinone intermediate that can be attacked by nearby nucleophiles, resulting in a stable cross-link. 4 Using as a model system a high affinity complex comprised of the yeast Gal80 protein and a DOPA-containing peptide mimicking the Gal4 activation domain, 5-7 we showed that this chemistry provided a high yield of cross-linked product when the purified components were mixed together in standard biochemical buffers or in crude cell extracts. The reaction was relatively rapid, reaching completion in less than one minute. Finally, control experiments demonstrated that no coupling occurred unless the DOPA-containing molecule was specifically bound to the protein receptor, suggesting that false positives will be rare using this chemistry.As DOPA appears to be a promising cross-linking agent with which to study peptide-protein and protein-protein interactions, it is important to understand the mechanism of cross-linking in more detail. While it seems clear that the critical intermediate is an ortho-quinone, it is less *Corresponding author. Thomas.Kodadek@utsouthwestern.edu. In this study, we report the results of experiments design...

Identification of Residue-to-residue Contact between a Peptide Ligand and Its G Protein-coupled Receptor Using Periodate-mediated Dihydroxyphenylalanine Cross-linking and Mass Spectrometry

“…The results suggested that most receptors (ϳ90%) of the wild type and K269H mutant were crosslinked, whereas about 25 and 75% of the K269A and K269C receptors, respectively, were cross-linked. It has previously been reported that the periodate-activated DOPA system often results in high yields (30). The residual amount of crosslinking found in the wild-type receptor in the presence of ␣-factor (Fig.…”

“…However, to efficiently couple to DOPA, the side groups of these amino acids and DOPA must be in very close proximity (30). Based on these findings, we investigated the cross-linking of Bio-DOPA 1 -␣-factor in Ste2p mutants K269A, K269C, and K269H that have lysine replaced with alanine, cysteine, and histidine, respectively.…”

“…MALDI-TOF spectra were acquired on a Bruker Daltonics Microflex using the reflector method. Masses were calculated using PROWL peptide mass prediction tools (29) and also based on the chemistry of the DOPA cross-linking (30).…”

Infiltration of chitin by protein coacervates defines the squid beak mechanical gradient

“…Therefore, we investigated the crosslinking potential of the DgHBPs with catechols using DgHBPpep. Sodium periodate (NaIO 4 ) was used to oxidize 4-methylcatechol (4MC) to the quinone form before crosslinking with deprotonated peptidyl His residues via Michael addition reactions 35 . Crosslinking was performed on both solution and coacervate forms of DgHBPpep (0.5 M ionic strength, pH 6 and 0.5 M ionic strength, pH 8, respectively) (Supplementary Fig.…”

Self-Assembly and Adhesion of DOPA-Modified Methacrylic Triblock Hydrogels

“…28,29 If the pH of the swelling solution is increased above pK aI , the DOPA groups become ionized, and they are no longer hydrophobic. This result is most clearly observed for the DOPA40 gel.…”