Design and NMR conformational study of a β-sheet peptide based on Betanova and WW domains

A good approach to test our current knowledge on formation of protein β-sheets is de novo protein design. To obtain a three-stranded β-sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed β-sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural β-sheet domain that is stable in absence of disulfide bridges. Some Betanova-LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt β-sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest β-sheet population according to the CD spectra, named 3SBWW-2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW-2 adopts a well defined three-stranded antiparallel β-sheet structure with a disordered C-terminal tail. To discern between the contributions to β-sheet stability of strand residues and the C-terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW-2 but lacking the C-terminal extension, named Betanova-LYYL, was also investigated. β-Sheet stability in these two peptides, in the parent Betanova-LLM and in WW-P, a prototype WW domain, decreased in the order WW-P > 3SBWW-2 > Betanova-LYYL > Betanova-LLM. Conclusions about the contributions to β-sheet stability were drawn by comparing structural properties of these four peptides. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 The Protein Society.