Design and solution structure of a well-folded stack of two beta-hairpins based on the amino-terminal fragment of human granulin A

Four amino acid substitutions were introduced into a peptide corresponding to the aminoterminal subdomain (30-31 residues) of human granulin A (HGA) i n order to assess the contributions of a hydrophobic framework and other in teractions to structure stabilization of the stack of two P-hairpins The re sulting hybrid peptide, HGA 1-31 (D1V, K3H, S9I, Q20P) with four free cyste ines, spontaneously formed a uniquely disulfide-bonded isomer with an expec ted [1-3, 2-4] disulfide pairing pattern. This peptide was characterized in detail by use of NMR and shown to assume a highly stable structure in solu tion, in contrast to the amino-terminal 1-30 fragment of human granulin A. The prototype peptide, or HGA 1-30 (C17S, C27S), had lower resistance to ch emical reduction and proteolysis, broad NH and Ha proton resonances, lower proton resonance dispersion, and no slowly exchanging amide protons. Two ot her peptides, HGA 1-30 (C17S Q20P, C27S) and HGA 1-31 (D1V, K3H, S9I, C17S, C27S), with either Pro20 stabilizing a potential reverse turn or with a hy drophobic cluster consisting of Val1, His3, and Ile9, had sharper and sligh tly better dispersed NH and Ha proton resonances, but still no slowly excha nging amide protons. The solution structure of HGA 1-31 (D1V, K3H, S9I, Q20 P) indicates that it adopts a well-folded conformation of a stack of two P- hairpins, as found for the amino-terminal subdomain of the prototypic carp granulin-l with representative P-hairpin stacks. These results highlight th e importance of both hydrophobic and turn-stabilizing interactions for the structural integrity of the hairpin stack scaffold. The conformational stab ility appears to be maintained by a combination of the well-formed second b eta-hairpin and two hydrophobic clusters, one located at the interface betw een the two beta-hairpins and the other on "top" of the first beta-hairpin. The implications of these findings for the design of conformationally stab le hairpin stacks are discussed.