Early Stages of Apomyogobin Folding

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This project is aimed at elucidating the early stages of folding of apomyoglobin (apoMb), an a-helical protein known to fold via a series of partially structured states. The structural and kinetic properties of the conformational changes associated with the transition from the unfolded state to an ensemble of intermediate states populated at acidic pH will be studied by combining continuous-flow fluorescence and quenched-flow H/D exchange measurements on the microsecond time scale with site-directed mutagenesis. Key residues involved in stabilizing early intermediates and transition states in folding will be identified by measuring the thermodynamic and kinetic consequences of alanine substitutions at conserved positions within the protein core. The formation of specific tertiary interactions during folding will be probed by replacing small residues at specific helix-helix docking sites by larger hydrophobic side chains, which will disrupt specific side chain contacts while maintaining non-specific hydrophobic interactions. Specific helix pairing interactions will be observed by using cysteine as an intramolecular quencher of tryptophan fluorescence. A novel quenched-flow hydrogen exchange experiment with a dead-time of 60 ms will provide complementary information on the acquisition of hydrogen-bonded structure during the initial stages of apoMb folding. The results obtained will identify critical residues and interactions (the folding nucleus) involved in directing structure formation during initial stages of folding, and will thus elucidate the sequence determinants of protein folding. The findings will provide a firm experimental framework for developing and testing theoretical and computational models of complex folding reactions involving intermediate states.Advanced rapid mixing techniques and detection methods developed in the principal investigator's laboratory not only represent a unique resource for the protein folding community, but also benefit other areas of research, such as kinetic studies of rapid conformational changes and enzymatic reaction mechanisms. This project offers extensive opportunities for training future scientists in experimental techniques and theoretical concepts, including protein biochemistry, advanced kinetic techniques, optical and NMR spectroscopy, and the principles of protein structure and folding. Although the Fox Chase Cancer Center is not a degree-granting institution, the principal investigator has access to and will train students at all levels, including a) graduate students from the University of Pennsylvania and Temple University; b) graduate students visiting Fox Chase through a partnership with universities in Russia; c) a summer research program for undergraduate students; and (d) the Howard Hughes Medical Institutes Student Scientist Program, which places gifted students from local high schools in Fox Chase labs.