Structure and Stabilities of Solution and Gas Phase\nProtein Complexes

Collision-induced\nunfolding (CIU) has provided new levels of understanding\nof the stabilities and structure(s) for gas phase protein and protein\ncomplex ions formed by electrospray ionization (ESI). Variable-temperature\n(vT-ESI) data provide complementary information about temperature-induced\nfolding/unfolding (TIU) reactions of solution phase ions. Results\nobtained by using CIU and TIU provide complementary information about\nstabilities of gas phase versus solution phase ions. Such comparisons\nmay provide the most direct experimental approach to answer a long-standing\nquestion from Fred McLafferty: “For how long, under what conditions,\nand to what extent, can solution structure be retained without solvent?”\nAnswers to this question require greater understanding of the (i)\nstructure(s), stabilities, and dynamics of proteins/protein complexes\nin solution prior to ESI; (ii) effects of water removal by droplet\nfission and “freeze-drying” by evaporation of water\nfrom the nanodroplet; and (iii) potential reactions and structural\nchanges that may occur as the ions traverse the heated capillary,\nthe final stage in the transition to solvent-free gas phase ions.\nHere, we employ vT-ESI coupled with ion mobility-mass spectrometry\n(IM-MS) as a means to provide more detailed answers to the above-mentioned\nquestions. Apo- and metalated-metallothionein-2A (MT), a cysteine-rich\nmetal binding protein, and various proteoforms of transthyretin (TTR),\na homotetrameric (56 kDa) retinol and thyroxine transporter protein\ncomplex were studied to examine distinct features of CIU and TIU across\ntwo different types of protein complexes. The results in this work\nshed light on the capabilities of CIU, TIU, and average charge state\n(Z<sub>avg</sub>) for probing the rugged energy landscape of native\nproteins and highlights the effects of water and cofactors (metal\nions) on the structure and stabilities of proteins and protein complexes.