Synthesis and NMR solution structure of an alpha-helical hairpin stapled with two disulfide bridges

Helical coiled-coils and bundles are some of the most common structural mot ifs found in proteins. Design and synthesis of alpha-helical motifs may pro vide interesting scaffolds that can be useful as host structures to display functional sites. thus allowing the engineering of novel functional minipr oteins. We have synthesized a 38-amino acid peptide. alpha(2)p8, encompassi ng the alpha-helical hairpin present in the structure of p8(MTCP1) as an al pha-helical scaffold particularly promising for its stability and permissiv eness of sequence mutations. The three-dimensional structure of this peptid e has been solved using homonuclear two-dimensional NMR techniques at 600 M Hz. After sequence specific assignment, a total of 285 distance and 29 dihe dral restraints were collected The solution structure of alpha(2)p8 is pres ented as a set of 30 DIANA structures, further refined by restrained molecu lar dynamics, using simulated annealing protocol with the AMBER force field . The RMSD values for the backbone and all heavy atoms are 0.65 +/- 0.25 an d 1.51 +/- 0.21 Angstrom, respectively. Excised from its protein context, t he alpha-hairpin keeps its native structure: an alpha-helical coiled-coil, similar to that found in superhelical structures, with two helices spanning residues 4-16 and 25-36. and linked by a short loop. This motif is stabili zed by two interhelical disulfide bridges and several hydrophobic interacti ons at the helix interface, leaving most of its solvent-exposed surface ava ilable for mutation. This alpha-helical hairpin, racily amenable to synthet ic chemistry and biological expression system, may represent a stable and v ersatile scaffold to display new functional sites and peptide libraries.