Protein-ligand binding analysis in complex mixtures using amide hydrogen exchange and MALDI mass spectrometry

Noncovalent interactions of proteins with oligonucleotides, small molecules, metal ions, peptides, and other proteins form the basis of many biological processes. These interactions are important for signaling, regulation of enzymatic activity, immune response processes, and many other functions in living systems. Thus, the thermodynamic analysis of protein-ligand interactions is important for understanding many important biological processes. Conventional approaches for such analyses have included calorimetric and spectroscopic-based methods. These approaches generally require large amounts of highly purified protein and they are not amenable to high-throughput analysis. Recently, an H/D exchange and mass spectrometry-based technique termed SUPREX, (Stability of Unpurified Proteins from Rates of H/D Exchange) was developed for the quantitative determination of protein folding free energy values (i.e., ΔGf values). The SUPREX technique affords several unique experimental advantages over conventional methods for making thermodynamic measurements on proteins and protein-ligand complexes. These advantages include speed, sensitivity, and the ability to analyze both highly purified protein samples as well as protein samples in complex biological mixtures such as those found in vivo.;This dissertation is focused on the application of the SUPREX technique to the thermodynamic analysis of protein-ligand complexes in complex mixtures. The three model systems in this work were studied in mixtures that included multiple proteins, cell lysates, and ex vivo membrane preparations. The first two model systems included the bacterial transport protein, ferric binding protein (FbpA) and the mammalian transport protein, transferrin (Tf). These proteins were studied to provide insight into the Fe3+ acquisition and transport mechanisms in mammals and microbes. The third model system was the protein LC8, which was studied to address the mechanistic basis of its function in the cytoplasmic dynein microtubule-based motor multi-protein complex.;A major part of this work involved the development and validation of new SUPREX protocols and data analysis methods for qualitative and quantitative thermodynamic measurements of protein-ligand binding. These protocols and data analysis methods were validated in vitro using known ligands to the FbpA and transferrin systems. Subsequent studies of ligand binding interactions to these two protein systems were then conducted in more complex biological mixtures to better understand their ligand binding properties in vivo. The work described on the LC8 system represents the first thermodynamic measurements of ligand binding to this system, as such measurements on multi-protein complexes are not easily performed using traditional calorimetric and/or spectroscopic methods.