Probing pH mediated protein-protein interactions via photocrosslinking

Abstract

Diverse protein-protein interactions are mediated by pH, a key parameter in dictating the charge and structure of nearly all proteins. Although photocrosslinking is powerful in trapping transient protein interactions in living cells, the pH-dependent protein interactions are exceedingly difficult to capture, largely due to the significant change of the protein binding interfaces upon pH variations. Here, we report that our newly developed alkyl diazirine-based unnatural amino acid photocrosslinker-DIZPK is capable of detecting the <italic>in vivo</italic> substrates of HdeA, a major acid-protection chaperone. Among the identified substrates of HdeA, we found two important periplasmic chaperone proteins: DegP and SurA, that were protected by HdeA at a low pH, but subsequently assist the HdeA-mediated recovery of other client proteins. This ATP-independent chaperone cooperation may enhance the acid resistance of enteric bacteria. Based on these results, we proposed a “chaperone cooperation” model in order to illustrate how acid-protection chaperones are employed by enteric bacterial to cope with acid stress. The highly adaptable photocrosslinker presented here can be generally applicable for surveying various pH-dependent protein-protein interactions in prokaryotic and eukaryotic cells.