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PEDS Advance Access published online on January 24, 2008
Protein Engineering Design and Selection, doi:10.1093/protein/gzm070
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Two-dimensional surface display of functional groups on a β-helical antifreeze protein scaffold

Maya Bar1, Tali Scherf2 and Deborah Fass1,3

1Department of Structural Biology 2Chemical Research Support, Weizmann Institute of Science, Rehovot 76100, Israel

3 To whom correspondence should be addressed. E-mail: deborah.fass{at}weizmann.ac.il

We tested a disulfide-rich antifreeze protein as a potential scaffold for design or selection of proteins with the capability of binding periodically organized surfaces. The natural antifreeze protein is a β-helix with a strikingly regular two-dimensional grid of threonine side chains on its ice-binding face. Amino acid substitutions were made on this face to replace blocks of native threonines with other amino acids spanning the range of β-sheet propensities. The variants, displaying arrays of distinct functional groups, were studied by mass spectrometry, reversed-phase high performance liquid chromatography, thiol reactivity and circular dichroism and NMR spectroscopies to assess their structures and stabilities relative to wild type. The mutants are well expressed in bacteria, despite the potential for mis-folding inherent in these 84-residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded β-helical scaffold, thermally stable and remarkably tolerant of amino acid substitutions, is therefore useful for design and engineering of macromolecules with the potential to bind various targeted ordered material surfaces.

Keywords: antifreeze protein/beta-helix/circular dichroism/disulfide bonds/protein folding

Received September 3, 2007; revised November 1, 2007; accepted November 5, 2007.


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