title: Design and solution structure of a well-folded stack of two beta-hairpins based on the amino-terminal fragment of human granulin A.
abstract: Four amino acid substitutions were introduced into a peptide corresponding to the amino-terminal subdomain (30-31 residues) of human granulin A (HGA) in order to assess the contributions of a hydrophobic framework and other interactions to structure stabilization of the stack of two beta-hairpins. The resulting hybrid peptide, HGA 1-31 (D1V, K3H, S9I, Q20P) with four free cysteines, spontaneously formed a uniquely disulfide-bonded isomer with an expected [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 solution, 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 chemical reduction and proteolysis, broad NH and H(alpha) proton resonances, lower proton resonance dispersion, and no slowly exchanging amide protons. Two other 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 hydrophobic cluster consisting of Val1, His3, and Ile9, had sharper and slightly better dispersed NH and H(alpha) proton resonances, but still no slowly exchanging amide protons. The solution structure of HGA 1-31 (D1V, K3H, S9I, Q20P) indicates that it adopts a well-folded conformation of a stack of two beta-hairpins, as found for the amino-terminal subdomain of the prototypic carp granulin-1 with representative beta-hairpin stacks. These results highlight the importance of both hydrophobic and turn-stabilizing interactions for the structural integrity of the hairpin stack scaffold. The conformational stability appears to be maintained by a combination of the well-formed second beta-hairpin and two hydrophobic clusters, one located at the interface between the two beta-hairpins and the other on "top" of the first beta-hairpin. The implications of these findings for the design of conformationally stable hairpin stacks are discussed.
authors.affiliation: Biomolecular NMR Laboratory and the Montreal Joint Centre for Structural Biology, Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec, Canada H4P 2R2.
authors.forename: D
authors.initials: D
authors.lastname: Tolkatchev
authors.forename: A
authors.initials: A
authors.lastname: Ng
authors.forename: W
authors.initials: W
authors.lastname: Vranken
authors.forename: F
authors.initials: F
authors.lastname: Ni
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002347:Carps; D004220:Disulfides / Q000737:chemistry; D000077155:Granulins; D006133:Growth Substances / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012996:Solutions
chemical_list: D004220:Disulfides; D000077155:Granulins; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D012996:Solutions