Stability of biomolecules and noncovalent complexes studied by nanoflow electrospray FT-ICR mass spectrometry

• Main Author: Daneshfar, Rambod
• Format: Dissertation
• Language: English
• Subjects: chemistry, analytical
• ISBN: 9780494139561; 0494139560

To understand the origin of the differential reactivity of nucleobases in oligodeoxynucleotides (ODNs), the loss of neutral nucleobases from a series of deprotonated thymine-rich ODN 10-, 15- and 20-mers, XT9, T 9X, T5XT4, XT14, XT19 and T19X, containing a single reactive base (X = adenine (A), guanine (G), cytosine (C)) were investigated. Time-resolved blackbody infrared radiative dissociation experiments, implemented with a Fourier transform ion cyclotron mass spectrometer (FT-ICR/MS) equipped with a nanoflow electrospray (nanoES) ion source, were used to evaluate the dissociation kinetics and energetics for the loss of a neutral nucleobase (XH). Over the temperature range investigated, the kinetics for the loss of CH was significantly slower than the loss of AB and GH. In contrast to loss of CH, the activation energies (E a) for the loss of AH are sensitive to the length and charge state of the ODN and position of the base. Importantly, the trend in Ea values does not correlate with trends in deprotonation enthalpies or proton affinities of the nucleobases which indicates that the differences in energetics are not simply a result of differences in their gas phase acidity or basicity. Instead, the results suggest an inherent difference in the internal solvation of the reactive base, with A being significantly more solvated than C.
A novel temperature-controlled nanoES device which allows for determination of protein-ligand binding affinities (Kassoc) over a range of temperatures was developed. Values of Kassoc for carbohydrate complexes of two carbohydrate binding proteins were measured for solutions in the temperatures range of 5--40°C. From the temperature dependence of MS-derived Kassoc values, the enthalpies and entropies of association were determined. The thermochemical data were found to be in good agreement with values determined previously by isothermal titration calorimetry.
The influence of solution conditions (protein concentration, temperature, pH, and ionic strength) on stability of structurally similar B5 homopentamers of Shiga toxins (Stx) 1 and 2 were investigated using temperature-controlled nanoES-FT-ICR/MS. At temperatures ranging from 0 to 60°C, the Stx1 B subunit exists almost exclusively in its pentameric form at neutral pH. Acidification was found to promote disassembly. The Stx2 subunit forms abundant pentamers at subunit concentrations > 50 muM, neutral pH, ambient temperature and relatively high salt concentration. The lower thermodynamic stability of the B subunits of Stx2, compared with Stx1, is a significant finding because it may provide an explanation for the greater toxicity of Stx2 relative to Stx1.