The prediction of the three dimensional structure of a globular protein from its amino acid sequence along with the elucidation of the mechanisms by which proteins fold represent extremely important unsolved problems of contemporary molecular biology. The overall objective of the proposed FIRCA project which is an extension of a NIH Program Project, No. 2 PO1- GM38794-O6 entitled, "Structural Basis of Protein Folding & Macromolecular Assembly" is the development of a methodology to predict the secondary, tertiary and quaternary structure along with the folding pathway of a number of globular proteins from the amino acid sequence alone. The method is based on a hierarchical approach to protein folding. First, high resolution lattice models of proteins comprised of an alpha-carbon plus reduced off lattice side chain description provide the overall folding pathway and folded conformations. The resulting lattice structures are estimated for the alpha-carbons to have a 2-4 Angstroms rms deviation from the corresponding backbone atoms of the experimentally determined native structure. These lattice structures will provide the set of predicted secondary structure and side chain contact constraints which are used in a molecular dynamics refinement protocol aimed at producing full atom structures. In particular, the following will be addressed in this proposal: (1). The methodology comprised of reduced, discretized models designed to fold single domain, monomeric globular proteins will be extended to treat multimeric proteins. Phenomenological interaction parameters will be derived to reflect relative preferences for interfacial interactions. (2). The methodology will be tested on series of four helix bundles designed by DeGrado and coworkers and composed of four short peptides, two longer peptides, and one long polypeptide, where the amino acid sequence of the helical regions is highly simplified and the same in all cases. (3). The folding of ROP dimer, whose native conformation is a four helix coiled coil bundle formed from the association of two helical hairpins, will be examined. The folding process and resulting quaternary structure will be compared to that obtained for the redesigned monomeric sequence. (4). The nature of the monomer-dimer equilibrium of avian pancreatic polypeptide and crambin will be explored. These molecules represent cases where both the isolated monomer and the multimeric form of the globular protein are stable. (5). The stability of glucagon as a function of the monomer-trimer equilibrium will be explored. This molecule adopts well defined conformations only on association to trimers or higher order multimers. (6). The folding process of insulin and proinsulin will be addressed. This represents the very important situation where the native conformation of a mature protein can be obtained only in the preprocessed form; i.e., the mature protein is in a metastable state.