Iterative protein redesign

An iterative redesign protocol for the transformation of a non-native pepti de into a series of nativelike proteins derived from elementary considerati ons of biological evolution coupled with H-1 NMR as an artificial selection criterion is presented. Each of three heptad d position leucines in the he lix-helix interfaces of the prototype heme protein maquette, [H10H24](2) or (alpha-SS-alpha)(2), were replaced in a unit modification per helix by mor e conformationally restricted beta-branched and aromatic amino acids. The s econdary structure content (evaluated by circular dichroism and infrared sp ectroscopies), solvent accessibility of the tryptophan residues (measured b y fluorescence spectroscopy), global stability (quantitated by isothermal c hemical denaturation), and degree of conformational specificity (determined by H-1 NMR spectroscopy) of the resultant peptides were determined. Improv ement in the degree of conformational specificity was utilized as a selecti on criterion to choose three of the nine singly modified peptides for a sec ond unit modification per helix. Five of the resultant seven doubly modifie d peptides were nativelike, as determined by NMR spectroscopy. One of the d oubly modified peptides was chosen for a third unit modification per helix, which resulted in three triple variants with low conformational specificit y. The 20 proteins synthesized fold into discrete, stable four-alpha-helix bundles but with differing stabilities (-Delta G(H2O) from 10.50 to 22.73 k cal/mol) and varying degrees of conformational specificity (multistructured to singular solution structure). The singly, doubly, and triply modified ( per helix) peptides can be mapped onto a contiguous segment of sequence spa ce, providing the first experimental map of this vast molecular terrain. Th e energetic contours of sequence space are revealed in terms of both global folding energies (-Delta G(H2O)) and degree of conformational specificity within the hydrophobic core. Remarkably, six of the peptides studied (30%) contain uniquely structured hydrophobic cores amenable for NMR structural d etermination. The map of sequence space readily identifies a plastic site w ithin the protein, a position which can be occupied by various amino acids with retention of a uniquely structured global fold, thereby providing a po ssible route for iterative redesign toward chemical enzymatic function.