Kinetic intermediates in the folding of gaseous protein ions characterizedby electron capture dissociation mass spectrometry

Alternative mechanisms propose that protein folding in solution proceeds ei ther through specific obligate intermediates or by a multiplicity of routes in a "folding funnel". These questions are examined in the gas phase by us ing a new method that provides details of the noncovalent binding of solven t-free protein ions. Capture of an electron by a multiply charged cation ca uses immediate dissociation (ECD) of a backbone bond, but with negligible e xcitation of noncovalent bonds; thus ECD of a linear protein ion produces t wo measurable fragment ions only if these are not held together by noncoval ent bonds. Thermal unfolding of 9+ ions of cytochrome c proceeds through th e separate unfolding of up to 13 backbone regions (represented by 44 bond c leavages) with melting temperatures of <26 to 140<degrees>C. An 0.25 s lase r IR pulse induces unfolding of 9+ ions in <4 s in six of these regions, fo llowed by their refolding in 2 min. However, for the 15+ ions a laser IR pu lse causes slower unfolding through poorly defined intermediates that leads to far more ECD products (63% increase in bond cleavages) after 1 min, eve n more than heating to 140<degrees>C, with refolding to a more compact conf ormation in 10 min. Random isomerization appears to produce a dynamic mixtu re of conformers that folds through a variety of pathways to the most stabl e conformer(s), consistent with a "folding funnel"; this might also be cons idered as an extension of the classical view to a system with a far smaller free energy change yielding multiple conformers. As cautions to inferring solution conformational structure from gas-phase data, no structural relati onship between these gaseous folding intermediates and those in solution is apparent, consistent with reduced hydrophobic bonding and increased electr ostatic repulsion. Further, equilibrium folding of gaseous ions can require minutes, and even momentary unfolding of an intermolecular complex during this time can be irreversible.