The proposed research concerns a number of experimental approaches designed to address the question of how the primary sequence of a protein influences spontaneous folding and stability. The investigations will initially involve variants of a protein, bacteriophage lysozyme. Two other bacteriophage proteins will eventually replace the lysozyme as the paradigm of protein folding. Several single- and multiple-site mutants of bacteriophage lysozyme will be selected from a group of existing variants and will be employed in studies of their thermodynamic stability. The types of mutants which will be used include those which, compared to the wild type protein, exhibit altered: (1) sensitivity to thermal inactivation, (2) numbers of charged residues, and (3) cysteine residues capable of forming disulfide bridges. An attempt will be made to experimentally determine electrostatic contributions to protein stability as a function of sequence. Proteins containing genetically-engineered disulfide bridges will be studied to determine the nature of the stability change introduced and also to obtain the free energy of bridge formation for each as a function of pH and temperature. From the latter and the free energy of denaturation of the bridged and un-bridged proteins, a thermodynamic cycle will be constructed. This will permit independent determination of how the free energy of the native and the denatured states are modified.
