|Abstract Number: ||260 - 4|
|Author Name: ||Charles McEwen - University of the Science|
|Session Title: ||Pittsburgh Conference Achievement Award|
|Event Type: ||Awards|
|Event Title: ||Protein Conformational Evolution from Solution to Gas Phase: Electrospray Ionization Yields Unfolding and New Folding|
|Presider Name:||Jane Chan|
|Affiliation:||Bechtel Bettis, Inc.|
|Date: ||Monday, March 18, 2013|
|Start Time: ||08:45 AM (Slot #4)|
|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 electron capture dissociation (ECD), IR or collisional activation (CA), H/D exchange (HDX), infrared photodissociation spectroscopy (IRPD), and charge site spectra. |
ECD spectra of ubiquitin 7+ ions 0.2 and 5 s after ESI are consistent with extensive unfolding in <0.2 s by two major processes, followed immediately by formation of new salt bridges, e.g., Asp-21-Arg-54 and Arg42-HOOCGly76 that are overlapping and E51-R54-D58. Charge site spectra show that both terminal regions become α-helical, 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 characterization; the postulated double overlap region of residues Arg42-Arg54 show the largest increases in H/D exchange after CA.
Ion cell storage for ≥40 s produces more extensively folded conformers; the least folded have no tertiary structure in terminal regions 1-10 and 73-76. After IR denaturation of these “mature” ions, refolding is characterized kinetically by ECD for 0.2 s to 10 min; surprisingly, the earliest (1 s) first-order folding is consistent with formation of the same two salt bridges observed for the native state 0.2 s after ESI.