A strategy for the de novo design of helical proteins with stable folds

This paper describes peptide analogs and the design strategythat were used to facilitate the final construction of a denovo-designed protein (ALIN) whose stable tertiary fold hasbeen determined recently by NMR spectroscopy. Previous studieshave suggested that the main problem in the de novo design ofproteins is the attainment of a protein with a defined fold.To effectively overcome this mainchain multiconformation problem,three related steps, with experimental evaluation of the designhypotheses for each step, were pursued in the design process.Firstly, 15-residue sequences with experimentally verified highhelicities were selected for the helical regions. Secondly,hydrophobic and electrostatic interhelical interactions as wellas an interhelical disulfide bridge were designed to favor anantiparallel configuration of the helix axis. Finally, a loopwith sufficient flexibility was inserted to stabilize the helicesin the desired orientation. To assess the design strategy, peptidescorresponding to each design step were synthesized and theirstructures verified experimentally by far-UV CD. As anticipated,ALIN was the most helical, and the SSbridged dimeric peptideswere more helical than their monomeric counterparts. The van'tHoff enthalpy change for ALIN computed from the CD denaturationcurve and assuming a two-state model was 50 kJ/mol, a valueclose to that observed for helical coiled-coils. Overall, thisreport shows that small, simple proteins can be built usingthe current knowledge of protein structures.