The overall goal of this proposal is to determine the mechanism by which the amino acid sequence of a protein directs the rapid and efficient folding to the native conformation. The approach is to use site-directed mutagenesis to identify sidechains which play key roles in folding. Those that have such roles will, when replaced, have discernable effects on the equilibrium and kinetic properties of folding. The protein to be studied is dihydrofolate reductase (DHFR) from Escherchia coli. DHFR is a relatively small monomeric protein which has no disulfide bonds or prosthetic groups and for which a high resolution X-ray structure exists. A combination of absorbance, fluorescence and circular dichroism spectroscopies have been used to establish a folding model for DHFR. This model postulates the existence of a number of transient folding intermediates whose structures and energetics will be studied. The rate limiting steps in folding which immediately precede the native conformation will also be subjects for further investigation. The information obtained from these studies will enhance the possibility of predicting the tertiary structure of a protein from its amino acid sequence, advance our understanding of the molecular defects in genetically transmitted diseases, and improve our ability to alter enzymes to improve catalytic properties.
