Study of Non-Covalent Protein-Hydrophobic Ligand Complexes by Electrospray Ionization Mass Spectrometry

This thesis describes the development and application of techniques based on electrospray ionization mass spectrometry (ES-MS) to quantify the protein-hydrophobic ligand interactions in vitro and to investigate the structure and stability of these complexes in the gas phase. Bovine β-lactoglobulin (Lg), and its interactions with fatty acids (FA) served as model systems for this study.;The stoichiometry and affinity of non-covalent hydrophobic interactions between Lg and six long chain FAs in solution were evaluated using the direct ES-MS assay. Under the optimized condition, binding constants (Ka) measured for the longer FAs are in good agreement with values obtained using a fluorescence assay. However, Ka values measured for shorter FAs are significantly smaller than expected; for the shortest FA investigated, no complex ions could be detected. These findings suggest the occurrence of in-source dissociation of the complexes. However, for the shorter FAs, affinities obtained using the reference ligand method were found in good agreement with reported values.;The structure and stability of the gaseous deprotonated ions of the (Lg + FA) complexes were investigated using various experimental and computational methods. Rate constants and the corresponding Arrhenius parameters were determined for the loss of neutral FA from the (Lg + FA)7- ions using time-resolved blackbody infrared radiative dissociation measurements. These kinetic data, together with results of molecular dynamics simulations provide compelling evidence that the acyl chain of FAs is retained within the hydrophobic cavity of Lg in the gas phase. Comparison of the dissociation kinetics for the gaseous (Lg + FA)n- ions with those determined by surface plasmon resonance spectroscopy for the complexes in water provides evidence for a late dissociative transition state for FA loss. A late dissociative transition state operating in the gas phase is also supported by results of deuterium kinetic isotope effects. Finally, the average energetic contributions of -CH3, >CH2 and -CH2=CH2- groups to the stability of the (Lg + FA)7- ions were established from the Arrhenius parameters determined for a series of saturated, unsaturated, and branched FAs.