Protein design with L- and D-alpha-amino acid structures as the alphabet.

Summarizing the implications of homochiral structures in interpeptide interactions, not only in the topology but also possibly in the physics of protein folding, this Account provides an overview of the concept of shape-specific protein design using D- and L-(alpha)amino acid structures as the alphabet. The molecular shapes accessible in de novo protein design are stereochemically defined. Indeed, the defining consideration for shape specificity in proteins to be alpha-helix/beta-sheet composites is the L configuration of the alpha-amino acid structures. The stereospecificity in shapes implies that protein shapes may be diversifiable stereochemically, that is, designable de novo, using D and L structures as the alphabet. Indeed, augmented with D enantiomers, Nature's alphabet will expand greatly in the diversity of polypeptide stereoisomers, for example, from 1(30) to 2(30)--that is, from one to ca. one billion--for a modestly sized 30-residue polypeptide. Furthermore, with each isomer having conformers stereospecific to its structure, molecular folds of specific shapes may be approachable sequentially when D and L structures are used as the alphabet. Illustrating the promise, 14-20-residue bracelet-, boat-, canoe-, and cup-shaped molecular folds were designed stereochemically or implemented as specific sequence plans in the D- and L-alpha-amino acid alphabet. In practical terms, canonical poly-L peptide folds were modified to the desired shapes via stereochemical mutations invoking enantiomer symmetries in the Ramachandran phi,psi space as the logic. For example, in designing the boat-shaped fold, the canonical beta-hairpin was reengineered in its flat planar structure via multiple coordinated L-to-D mutations in its position specific cross-strand neighbor residues, upturning its ends enclosing six side chains in a molecular cleft. While affirming the generality of the approach, the 20-residue molecular canoe and the 14-residue molecular cup are also presented as examples of the scope of functional design. The canoe, possessing alkali cation-specific catgrips in its main chain, and the cup, featuring an organic cation-specific aromatic triad in its side chains, do indeed display desired specificities in their ligand binding. Stereochemistry is, therefore, the crucial specifier of protein shapes and valuable as the tool for shape-specific protein design. Proteins in general, whether poly-L or mixed-D,L, require sequence effects of amino acid side chain structures for their stability, if not also for specifying them conformationally. The principles underlying these phenomena remain a puzzle, but studies invoking a stereochemical mutation approach to the problem have suggested that the poly-L structure may be crucial to the principles of sequential encoding of protein structures in amino acid side chains as the alphabet.