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

¹ European Molecular Biology Laboratory, Heidelberg D-69117, Germany
² Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, Bellaterra, Barcelona 08193, Spain
³ Instituto de Química-Física Rocasolano, Consejo Superior de Investigaciones Científicas, Madrid 28006, Spain

(RECEIVED February 28, 2006; FINAL REVISION May 25, 2006; ACCEPTED July 7, 2006)

A good approach to test our current knowledge on formation of protein -sheets is de novo protein design. To obtain a three-stranded -sheet mini-protein, we have built a series of chimeric peptides by taking as a template a previously designed -sheet peptide, Betanova-LLM, and incorporating N- and/or C-terminal extensions taken from WW domains, the smallest natural -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 -sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest -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 -sheet structure with a disordered C-terminal tail. To discern between the contributions to -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. -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 -sheet stability were drawn by comparing structural properties of these four peptides.

Keywords: antiparallel -sheet; NMR; peptide design; peptide structure; WW domain

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