Development and Application of Covalent-Labeling Strategies for the Large-Scale Thermodynamic Analysis of Protein Folding and Ligand Binding

Thermodynamic stability measurements on proteins and protein-ligand complexes can offer insights not only into the fundamental properties of protein folding reactions and protein functions, but also into the development of protein-directed therapeutic agents to combat disease. Conventional calorimetric or spectroscopic approaches for measuring protein stability typically require large amounts of purified protein. This requirement has precluded their use in proteomic applications. Stability of Proteins from Rates of Oxidation (SPROX) is a recently developed mass spectrometry-based approach for proteome-wide thermodynamic stability analysis. Since the proteomic coverage of SPROX is fundamentally limited by the detection of methionine-containing peptides, the use of tryptophan-containing peptides was investigated in this dissertation. A new SPROX-like protocol was developed that measured protein folding free energies using the denaturant dependence of the rate at which globally protected tryptophan and methionine residues are modified with dimethyl (2-hydroxyl-5-nitrobenzyl) sulfonium bromide and hydrogen peroxide, respectively. This so-called Hybrid protocol was applied to proteins in yeast and MCF-7 cell lysates and achieved a ~50% increase in proteomic coverage compared to probing only methionine-containing peptides. Subsequently, the Hybrid protocol was successfully utilized to identify and quantify both known and novel protein-ligand interactions in cell lysates. The ligands under study included the well-known Hsp90 inhibitor geldanamycin and the less well-understood omeprazole sulfide that inhibits liver-stage malaria. In addition to protein-small molecule interactions, protein-protein interactions involving Puf6 were investigated using the SPROX technique in comparative thermodynamic analyses performed on wild-type and Puf6-deletion yeast strains. A total of 39 proteins were detected as Puf6 targets and 36 of these targets were previously unknown to interact with Puf6. Finally, to facilitate the SPROX/Hybrid data analysis process and minimize human errors, a Bayesian algorithm was developed for transition midpoint assignment. In summary, the work in this dissertation expanded the scope of SPROX and evaluated the use of SPROX/Hybrid protocols for characterizing protein-ligand interactions in complex biological mixtures.

Coordinated analysis of delayed sprites with high-speed images and remote electromagnetic fields

Simultaneous measurements of high-altitude optical emissions and magnetic fields produced by sprite-associated lightning discharges enable a close examination of the link between low-altitude lightning processes and high-altitude sprite processes. We report results of the coordinated analysis of high-speed sprite video and wideband magnetic field measurements recorded simultaneously at Yucca Ridge Field Station and Duke University. From June to August 2005, sprites were detected following 67 lightning strokes, all of which had positive polarity. Our data showed that 46% of the 83 discrete sprite events in these sequences initiated more than 10 ms after the lightning return stroke, and we focus on these delayed sprites in this work. All delayed sprites were preceded by continuing current moments that averaged at least 11 kA km between the return stroke and sprites. The total lightning charge moment change at sprite initiation varied from 600 to 18,600 C km, and the minimum value to initiate long-delayed sprites ranged from 600 for 15 ms delay to 2000 C km for more than 120 ms delay. We numerically simulated electric fields at altitudes above these lightning discharges and found that the maximum normalized electric fields are essentially the same as fields that produce short-delayed sprites. Both estimated and simulation-predicted sprite initiation altitudes indicate that long-delayed sprites generally initiate around 5 km lower than short-delayed sprites. The simulation results also reveal that slow (5-20 ms) intensifications in continuing current can play a major role in initiating delayed sprites. Copyright 2008 by the American Geophysical Union.

Systematic characterization of thermodynamic and dynamical phase behavior in systems with short-ranged attraction

In this paper we demonstrate the feasibility and utility of an augmented version of the Gibbs ensemble Monte Carlo method for computing the phase behavior of systems with strong, extremely short-ranged attractions. For generic potential shapes, this approach allows for the investigation of narrower attractive widths than those previously reported. Direct comparison to previous self-consistent Ornstein-Zernike approximation calculations is made. A preliminary investigation of out-of-equilibrium behavior is also performed. Our results suggest that the recent observations of stable cluster phases in systems without long-ranged repulsions are intimately related to gas-crystal and metastable gas-liquid phase separation.