Figure 2.
Partial unfolding in human Hck SH3 as monitored by HX MS. (A) Transformed mass spectra (from Ref. [1]) of Hck SH3 after various times in D2O from 1 minute to 8 hours [including undeuterated (0%) and maximally deuterated (100%) controls]. A bimodal pattern after 22 minutes of labeling is accentuated with two uniform Gaussian distributions: the lower-mass distribution is indicated with a green arrow and represents molecules that have not yet unfolded and become deuterated; the higher-mass distribution is indicated with a red arrow and represents molecules that have undergone partial cooperative unfolding and been labeled with deuterium. These mass spectra were acquired with instrument resolution of ~2,500, which is low relative to more recent data such as in Figure 6 which were obtained with nearly ~10,000 resolution. (B) The area of the lower-mass distribution (indicated with green) of an EX1 pattern is used to monitor the disappearance of the folded/undeuterated species along the time course of the labeling experiment. (C) The rate constant of unfolding and the unfolding half-life for an EX1 event can be determined with the equation shown. The slope of the line fit to the data yields the rate constant. In this example, partial unfolding in Hck SH3 was monitored at the times shown and compared to unfolding in the Hck SH3 domain when bound to the HIV Nef peptide (see [1] and main text for details).