The purpose of this proposal is to better understand the interrelationships of protein structure, stability, and dynamics using T4 lysozyme as a model. This moderate sized protein of 164 residues has a known 3-dimensional crystal structure. The protein has two structural domains and no natural disulfide bonds. Many mutants exist that modulate its thermodynamic stability and more than 100 mutant crystal structures have been determined by Brian Matthews' laboratory at the University of Oregon. We have assigned the amide resonances of the wild type and several mutant proteins using modern nuclear magnetic resonance techniques (NMR). We propose to use these methods to investigate (1) the ionization behavior of the 19 carboxyl groups of the protein with the goal of understanding electrostatic and polarity effects in the protein; (2) the dynamics of the folded state; (3) determine the role in the folding pathway of an early folding intermediate which was detected as a result of the protection to hydrogen exchange it afforded a limited set of amides; (4) complete the assignments of the sidechain resonances with the goal of determining the solution structure of the protein. %%% The linear amino acid sequence of a protein determines the complex three-dimensional shape that protein acquires in solution. This proposal investigates how that linear information is used to fold the protein into its three-dimensional structure. We will investigate: (1) the role of charge-charge interactions in stabilizing the folded state, (2) the rigidity of the folded state, (3) the time course of the conversion of a random, unfolded polymer chain into the final folded state, and (4) the structure, in solution, of the final folded state.
