Temporal Development of Protein Structure during S100A11 Folding and Dimerization Probed by Oxidative Labeling and Mass Spectrometry

Early Hydrophobic Collapse of α1-Antitrypsin Facilitates Formation of a Metastable State: Insights from Oxidative Labeling and Mass Spectrometry

“…48 Mixing efficiency and reproducibility of labeling were confirmed as described previously. 39 In addition, the mixer performance was verified optically in pH jump experiments on bromocresol purple in the presence of GdnHCl. 5 For reaction times of 2 min and longer, manual mixing was used to initiate folding, but the same continuous-flow setup as above was employed for oxidative labeling.…”

“…Kinetic folding experiments with oxidative labeling were performed using a twosyringe continuous-flow mixing device similar to that employed for a previous work. 39 For the 0.5-s and 7-s time points, syringes 1 and 2 were advanced at 2.5 and 47.5 μL min −1 , respectively, using a syringe pump (Harvard Apparatus, Boston, MA). Syringe 1 contained 200 μM α 1 AT denatured in 6 M GdnHCl, 10 mM phosphate buffer (pH 7.8), and 50 mM NaCl.…”

“…The digests were lyophilized and resuspended in 50 μL of water containing 0.5 μM bradykinin as an internal intensity standard. 39,52 Non-irradiated control samples were collected for every measurement by using the same solution conditions and mixing sequence, except that the laser was switched off. It is noted that neither trypsin nor chymotrypsin is strongly inhibited by α 1 AT.…”

“…38,39 The experiments discussed below were designed to mirror the recent HDX study of Tsutsui et al 30 HDX primarily reports on backbone hydrogen bonding, 5,40 whereas ·OH labeling measures side-chain accessibility. 34,38,39 Hydrophobic collapse causes solvent exclusion, thereby greatly contributing to protection from ·OH attack. 41 Thus, a side-by-side comparison of data obtained by the two methods allows an indepth examination of the α 1 AT folding mechanism.…”

An Improved Rapid Mixing Device for Time‐Resolved Electrospray Mass Spectrometry Measurements

“…[12] Various biological processes were characterized using these devices including protein folding and unfolding reactions,[13] enzymatic catalysis[14] and subunit association. [15]…”