Engineering of betabellin 15D: Copper(II)-induced folding of a fibrillar β-sandwich protein

Abstract

The inverse protein-folding problem has been explored by designing de novo the betabellin target structure (a 64-residue β-sandwich protein), synthesizing a 32-residue peptide chain (HSLTAKIpkLTFSIAphTYTCAVpkYTAKVSH, where p = DPro, k = DLys, and h = DHis) that might fold into this structure, and studying how its disulfide-bridged form (betabellin 15D) folds in 10 mM ammonium acetate with and without Cu2+. Circular dichroic spectropolarimetry indicated that at pH 5.8, 6.4, or 6.7 betabellin 15D exhibited β-sheet structure in the presence of Cu2+ but not in its absence. Electrospray mass spectrometry demonstrated that at pH 6.3 each molecule of betabellin 15D bound one or two Cu(II) ions. Electron microscopy showed that at pH 6.7 betabellin 15D formed short broad fibrils in the presence of Cu2+ but not in its absence. The observed width of the fibrils (7 ± 2 nm) was consistent with the width (6.8 nm) of a structural model of a fibril that contained two adjacent rows of betabellin 15D β-sandwiches joined lengthwise by multiple intersheet hydrogen bonds and widthwise by multiple Cu(II)-imidazole bonds. Electron paramagnetic resonance spectrometry revealed that some pairs of Cu(II) ions in a Cu(II)/betabellin 15D complex were magnetically coupled, which is consistent with the structural model of the Cu(II)/betabellin 15D fibril.