Electrospray ionization (ESI) of a singular protein tertiary structure in solution can produce ions of many charges, each of which can exist as multiple conformers. In contrast to accepted knowledge on solution behavior, however, there is no overall picture, even few structural details, on how gas phase unfolding and folding of the native conformer can produce these modifications. For transfer into the gas phase of the extensively-studied native ubiquitin ions, we can now report substantial details of near-complete conformational evolution using a combination of techniques such as electron capture dissociation (ECD), IR or collisional activation (CA), H/D exchange (HDX), and infrared photodissociation spectroscopy (IRPD). ESI of native ubiquitin yields conformationally unstable 7+ ions; pioneering studies with ion mobility spectroscopy by the Clemmer group have shown that possibly a dozen conformers are produced that have up to 65% increases in cross section. Our ECD spectra 0.2 and 5 s after ESI are consistent with extensive unfolding in <0.2 s by two major processes, separation of regions I and IV and then regions II and III. This is followed immediately by formation of new salt bridges, e.g., Asp-21-Arg-54 and Arg42-HOOCGly76 that are overlapping, and by E51-R54-D58. "Charge site (CS)" spectra, a new technique just developed by us, show that both terminal regions become α-helical also, consistent with conclusions of our 2002 ECD studies. In fact, amide N+-H absorptions at 3325 ±25 cm-1 consistent with charge-stabilized helical structures dominate (S/N ～ 200) the 3025-3775 cm-1 IRPD spectra of 7+ to 11+ gaseous ubiquitin ions. CA dissociation of the salt bridges provides confirmatory evidence of their location; the postulated double overlap region of residues Arg42-Arg54 is supported by the largest increases in H/D exchange after CA. Ion cell storage for ≥40 s at 25 °C produces more extensively folded conformers; the least folded have no tertiary structure in terminal regions 1_10 and 73-76. After IR laser denaturation of these "mature" ions, refolding is characterized kinetically by ECD for 0.07 s to 10 min; surprisingly, the earliest (1 s) first-order folding that stops cleavage at sites 24, 51, and 54 is consistent with formation of the same Asp-21-Arg-54 salt bridge observed for the native state 0.2 s after ESI. Far slower product decreases, even 10 min, from sites 11_17 and 57-65 indicate terminal region folding, while site 48 products that are little affected by the laser IR must represent a substantially different conformer. Although the singular native conformer in solution has produced a variety of different conformers in the gas phase, their basic four regions do relate directly to the β-sheets and helices of the original conformer.
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