Min-21 and Min-23, the smallest peptides that fold like a cystine-stabilized beta-sheet motif: Design, solution structure, and thermal stability

Small disulfide-rich proteins provide examples of simple and stable scaffol ds for design purposes. The cystine-stabilized beta-sheet (CSB) motif is on e such elementary structural motif and is found in many protein families wi th no evolutionary relationships. In this paper, we present NMR structural studies and stability measurements of two short peptides of 21 and 23 resid ues that correspond to the isolated CSB motif taken from a 28-residue squas h trypsin inhibitor. The two peptides contain two disulfide bridges instead of three for the parent protein, but were shown to fold in a native-like f ashion, indicating that the CSB motif can be considered an autonomous foldi ng unit. The 23-residue peptide was truncated at the N-terminus. It has a w ell-defined conformation close to that of the parent squash inhibitor, and although less stable than the native protein, it still exhibits a high T-m of about 100 degrees C. We suggest that this peptide is a very good startin g building block for engineering new bioactive molecules by grafting differ ent active or recognition sites onto it. The 21-residue peptide was further shortened by removing two residues in the loop connecting the second and t hird cysteines. This peptide exhibited a less well-defined conformation and is less stable by about 1 kcal mol(-1), but it might be useful if a higher flexibility is desired. The lower stability of the 21-residue peptide is s upposed to result from inadequate lengths of segments connecting the first three cysteines, thus providing new insights into the structural determinan ts of the CSB motif.