Proteins can now be constructed with any desired amino acid sequence. The potential applications of this technology in health and other areas are almost unlimited. Consequently, it is of upmost importance that we learn to predict how a protein will fold given just the amino acid sequence and that we learn how changes in the amino acid sequence will effect the function and stability of a protein. To this end, we propose to study the energetics and mechanism of folding of ribonuclease T1 (RNase T1) in depth. RNase T1 is an excellent model for protein folding studies. It is the smallest enzyme known with just 104 residues and folds to a compact globular conformation in which the hydrophobic core is sandwiched between a 4.5 turn alpha-helix and a 4 strand antiparallel beta-pleated sheet. The attactive features for the research described here are: (1) RNase T1 can be prepared with 0, 1, or 2 intact disulfide bonds and all of these will fold to give an enzymically active globular conformation, and (2) The conformational stability can be increased by over 4 Kcal/mole through changes in the NaC1 concentration. We plan to use established kinetic methods to investigate the mechanism of folding of RNase T1 with 0. 1, and 2 intact disulfide bonds, and over a range of conformational stabilities. Fluorescence, difference spectroscopy, rates of amide proton exchange, and activity mesurements will be used to follow folding. Key questions we hope to answer are: (1) How does the number of slow folding species and the number of structural intermediates depend on the number of intact disulfide bonds? (2) How does the number and concentration of structural intermediates depend on the conformational stability of the native protein? (3) Does the alpha-h elix or the Beta-sheet form first in the early stages of folding? The conformational stability and thermodynamics of folding of RNase T1 with 0, 1, or 2 intact disulfide bonds is being investigated using differential scanning calorimetry (in collaboration with Dr. Julian Sturtevant), and through an analysis of urea and guanidinium chloride denaturation curves. This should lead to a better understanding of the methods used to estimate conformational stability, and of the contribution of disulfide bonds to the conformational stability of proteins. By studying the effects of NaC1 and other salts on unfolding, we hope to learn the mechanism by which salts cause their remarkable stabilization of RNase T1. This may give us insight into the contribution of electrostatic interactions to the conformational stability of globular proteins.