DESCRIPTION: The overall goal of the proposed research is to design and produce libraries of de novo proteins that fold into well ordered alpha-helical structures. This goal will be pursued through an approach that uses both rational design and combinatorial methods. The first step will entail the design of a new structural scaffold specifying a uniquely folded 4-helix bundle. Each sequence position will be defined to occur in a particular environment in the desired alpha-helical structure (exposed vs. buried; alpha-helix vs. turn etc.). The second step will use combinatorial methods to generate a library of de novo amino acid sequences consistent with the designed scaffold. The combinatorial diversity will not be random, but instead, will be designed to deliver at each position only those amino acids most compatible with the structural environment of that position in the scaffold. The specific aims of this project are (1) to design a new structural scaffold that specifies a 4-helix bundle; (2) to design and construct a library of synthetic genes that encode a large collection of protein sequences consistent with this new scaffold; (3) to express and purify de novo proteins from this collection; (4) to biophysically characterize the structural and thermodynamic properties of the purified proteins and thereby assess whether they form molten globule ensembles or uniquely folded structures; and (5) to determine the 3-dimensional structures of representative proteins by NMR spectroscopy. The ability to design and construct large collections of uniquely folded de novo proteins will have a significant impact on biotechnology and medicine. Whereas current applications of biotechnology typically focus on the control, modification, and production of naturally occurring genes and proteins, future applications will not be limited to macromolecules provided by nature. The ability to produce libraries of well folded de novo proteins is an initial and essential step towards the ultimate goal of discovering novel proteins "tailor made" for applications in industry and medicine.