An experimental investigation of the stability and determinants of tertiary interactions in globular proteins is proposed. The helical protein myoglobin (Mb) is chosen for detailed study, because it crystallizes readily, its structure has been determined to high resolution, and the function, dynamics and thermodynamics of he molecule have been analyzed intensively. Variant peptides and proteins will be produced by: 1. site-directed mutagenesis of a cloned gene for sperm whale Mb in the plasmid pMB 413, in collaboration with Dr. S. Sligar and 2. solid state synthesis of fragments corresponding to the C-terminal 22 residues of the chain. The latter fragment from the H helix results from CNBr cleavage of Mb, and is helical in solution. We find it can be relegated with the residual 1-131 fragments to yield a native-like Mb containing two homoserines at sties 55 and 131. The spectroscopic, liganding, folding and NMR spectral properties of the synthetic fragments-MbC3 -and mutant Mb's will be determined. We will attempt to crystallize the mutants molecules, starting with Kendrew's standard ammonium sulfate conditions for sperm whale Mb, and compare the structure of these with the """"""""wild-type"""""""" species by X-ray crystallographic methods. A parallel analysis of the structural differences among peptides and mutant proteins will be initiated using 2D NMR spectroscopy. In this way, we hope to answer two important questions about protein folding. First, what is the relationship, if any, between the stability of an isolated helical fragment from a protein such as myoglobin to that of the intact protein? Second, can one establish structural, dynamic and energetic requirements for packing alpha helices within a protein matrix? Specifically, the tertiary interactions controlling three of the H helix subdomain packings will be analyzed, using structural data to characterize the local folding and changes in this folding in this folding on the one hand, and spectroscopic and thermodynamic data to define the corresponding changes in the function, stability, energetics and dynamics of the proteins on the other.
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