Engineering of betabellin 14D: Disulfide‐induced folding of a β‐sheet protein

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Abstract
The betabellin target structure consists of 2 32‐residue β sheets packed against each other by hydrophobic interactions. We have designed, chemically synthesized, and biophysically characterized betabellin 14S, a single chain, and betabellin 14D, the disulfide‐bridged double chain. The 32‐residue nongenetic betabellin‐14 chain (HSLTASIkaLTIHVQakTATCQVkaYTVHISE, a = D‐Ala, k = D‐Lys) has a palindromic pattern of polar (p), nonpolar (n), end (e), and β‐turn (t, r) residues (epnpnpnttnpnpnprrpnpnpnttnpnpnpe). Each half contains the same 14‐residue palindromic pattern (underlined). Pairs of D‐amino acid residues are used to favor formation of inverse‐common (type‐I') β turns. In water at pH 6.5, the single chain of betabellin 14S is not folded, but the disulfide‐linked betabellin 14D is folded into a stable β‐sheet structure. Thus, folding of the covalent dimer betabellin 14D is induced by formation of the single interchain disulfide bond. The binary pattern of alternating polar and nonpolar residues of its β sheets is not sufficient to induce folding. Betabellin 14D is a very water‐soluble (10 mg/mL), small (64 residues), nongenetic (12 D residues) β‐sheet protein with properties (well‐dispersed proton NMR resonances; Tm = 58 °C and δHm = 106 kcal/mol at pH 5.5) like those of a native protein structure.