Development Of Novel Carboxyl-reactive And Amine-reactive Cross-linkers And Their Application In Mass Spectrometric Analysis Of Protein Structures

Chemical cross-linking of proteins coupled with mass spectrometry analysis(CXMS)has become an established method to study structures of proteins and protein-protein interctions.It is less time-consuming and less demanding on sample purity than traditional methods,and it can map the interface between interaction proteins,so it is greeted with increasing popularity.Development of novel chemical cross-linkers has always been a driving force of CXMS,and it is the goal of this thesis.To this end,through collaborations with chemists,I have developed two series of novel cross-linkers:one is carboxylate-reactive diazo-containing cross-linkers(Diazoker)and the other is amine-reactive Di-Ortho-Phthalaldehyde(DOPA).The most widely used cross-linkers in CXMS have been limited to amine-reactive NHS spelled in full(NHS)ester cross-linkers.Since they target primarily lysine residues and protein N-termini,only a small portion of the structural information is retrieved.Chemical cross-linkers targeting the acidic residues glutamate(Glu)and aspartate(Asp)could greatly enhance the power of CXMS,as they constitute 12.2%of all amino acids in proteins and most of them are distributed on protein surfaces and protein-protein interacting regions.However,it has been difficult to develop chemistries that offer selectivity and efficiency under physiological conditions owing to the weak nucleophilic ability of carboxyl groups in aqueous solution.Here,we report a class of carboxylate-selective diazo-containing cross-linkers,which we call Diazokers(or collectively,Diazoker).Unlike previously developed carboxylate-selective cross-linkers such as pimelic acid dihydrazide(PDH),Diazoker does not require a coupling reagent,and has good ractivity and selectivity under near physiological conditions.After extensive optimization,I chose Diazoker 1,which has a 15.6-A polyethyleneglycol spacer arm,as the representative member.Tested on nine model proteins,Diazoker 1 generated on average 73 cross-linked peptide pairs per protein,and the Diazoker 1 cross-links have a higher compatibility rate with protein crystal structures than PDH cross-links.From a more complex protein mixture,Diazoker 1 and PDH identified 75 and 76 cross-linked peptide pairs,respectively.The Asp/Glu residues cross-linked by Diazoker 1 are not the same as those cross-linked by PDH;Diazoker 1 seems to favor acidic residues that are somewhat less exposed to solvent.In conclusion,Diazoker 1 is complementary to existing cross,linkers and expands the toolkit of CXMS for structural analysis of proteins.Like the commonly used NHS ester cross-linkers such as DSS or BS3,DOPA selectively reacts with lysine residues under near physiological conditions.However,DOPA has two distinctive advantages:it is non-hydrolyzable and has a faster reaction rate.Using HPr/EIN and EIIA/EIIB,two heterodimers of weakly interacting subunits,I demonstrated that DOPA2 can fix weak or transient protein-protein interactions better than DSS.I also found that DOPA works well even at low temperature or low pH,or in the presence of high concentrations of denaturants such as 8 M urea or 6 M guanidine hydrochloride.These properties enabled DOPA2 to capture structural changes of proteins during the unfolding process,as demonstrated using model protein RNase A.Therefore,DOPA not only provides a powerful tool for CXMS to identify weak protein-protein interactions,but also introduces CXMS to new application areas such as the studies of folding intermediates during protein folding or unfolding.