UC Berkeley

Methods for measuring ultrafast (1 µs) protein folding reactions, for forming highly charged protein ions, and for measuring the sizes and shapes of gas-phase protein ions are presented in this dissertation. Ultrafast protein folding is measured using rapid mixing from theta-glass emitters (double-barrel electrospray ionization emitters), in which reagent solutions are loaded into the different barrels of the emitters and rapid mixing occurs during electrospray ionization (ESI). Acidified aqueous solutions containing unfolded proteins are mixed with buffered aqueous solutions using the theta-glass emitters in order to increase the solution pH and induce protein folding during ESI. Reaction times in these experiments are obtained from the extent of folding that occurs and from folding time constants of model proteins. A 1.0 µs reaction time is achieved in these experiments, whereas only reactions occurring in 8 µs or greater could be monitored using rapid mixing techniques previously. A 2.2 µs folding time constant for the formation of a β-hairpin in a 14 residue peptide and the 1.5 and <0.4 µs folding time constants for the formation of polyproline II helix structures in 21 and 16 residue peptides, respectively, are obtained. To the best of our knowledge, these are the fastest folding events that have been directly measured using a rapid mixing device. In the second method presented here, highly charged protein ions are formed by ESI simply by using emitters with submicron outer diameter tips rather than the micron or larger outer diameter tips typical used for ESI. Increased charging is obtained with the submicron outer diameter tips for proteins that are positively charged in solution as a result of Coulombic attraction between the positively charged protein molecules in solution and the negatively charged glass surfaces in the tips of the emitters, which results in protein unfolding occurring prior to ESI. Using submicron outer diameter tips is a simple way to obtain highly charged protein ions with ESI that does not require exposing the proteins to additional chemicals. The final method presented here is for relating the drift times of ions in travelling wave ion mobility spectrometry (TWIMS) with collisional cross sections using computational simulations. Collisional cross sections obtained using this method with gentle instrument conditions are very similar to those obtained using static drift ion mobility spectrometry (average difference = 0.3%), demonstrating for the first time that collisional cross sections can be obtained from single TWIMS drift time measurements.