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Study of selected proteins and noncovalent protein-protein complexes by nano- and electrospray ionization tandem mass spectrometry Nesati, Victor Joseph

Abstract

This thesis summarizes the results of the investigation of conformational changes of individual proteins under denaturing/unfolding conditions by electrospray ionization mass spectrometry and an assessment of the stability of gas phase noncovalent protein-protein complexes by tandem mass spectrometry. The experiments studying changes in protein conformations utilized two relatively small, very stable proteins with a well characterized native structure, bovine pancreatic trypsin inhibitor (BPTI) and ubiquitin. Unfolding experiments were performed using changes in pH, and the addition of organic solvents. The results of the experiments confirmed the known resistance of these two proteins to strongly denaturing conditions. Proteins that are stabilized by multiple disulphide bonds can be unfolded by simply reducing the disulfides. BPTI contains three disulphide bonds, which can be removed by reduction with DTT. In addition, mutants with one or two native or non-native disulphide bonds can be prepared by different techniques. The contribution of the disulphide bonds to the folding of gas-phase BPTI was assessed using cross sections measured by an ion ertergy loss method. The charge states produced in ESI show that reduced BPTI is partially unfolded under the solution conditions used here. Collision cross sections show that removing the disulphide bonds causes BPTI to unfold in the gas phase but the increase in size is rather small (-17% in area). The results show that the disulphide bonds of BPTI contribute to the folding of the gas phase ion but that even in the absence of disulphide bonds, the protein maintains a compact structure. The experiments on evaluation of the stability of noncovalent interactions in the gas-phase utilized complexes between BPTI and tryptic proteins (trypsin, chymotrypsin, and trypsinogen). Trypsin, chymotrypsin and trypsinogen have similar molecular weights but different binding energies for BPTI in solution thus forming a system of natural mutants which become an attractive model for studying protein-protein interactions. Experiments were performed using electro- and nanospray ionization sources. The results of tandem mass spectrometry experiments were analyzed using a recently proposed collision model. It was found that the relative binding energies of the protein-protein complexes in the gas-phase do not correlate with their stability in solution.

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