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

Protein Engineering Design and Selection, Feb 2008

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.

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

Maya Bar 2 Tali Scherf 1 Deborah Fass 2 0 The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions , please 1 Chemical Research Support, Weizmann Institute of Science , Rehovot 76100, Israel 2 Department of Structural Biology 3To whom correspondence should be addressed. E-mail: 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 b-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 b-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 84residue proteins with 16 cysteines. We demonstrate that most of the mutants essentially retain the native fold. This disulfide bonded b-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. - An aesthetic and captivating example of the relationship between protein structure and function is provided by the antifreeze or thermal hysteresis proteins (THPs) (Davies et al., 2002). In particular, the b-helical insect THPs display a highly ordered two-dimensional (2D) array of threonine residues, in which the positioning of the side chain hydroxyl groups mimics the spacing of oxygen atoms on various facets of ice crystals (Liou et al., 2000b). The THP icebinding face, evolved for recognition of an inorganic crystalline material, differs from typical protein surfaces engaged in interactions with target molecules. When the targets are aperiodic macromolecules or small ligands, the binding surface presented by the protein is correspondingly rugged and varied. In contrast, the b-helical THP surface is broad, repetitive and flat. Interactions of proteins with crystalline surfaces have been observed in biomineralization and several pathological conditions (Perl-Treves and Addadi, 1988; Mann et al., 1989; Giachelli, 2005; Gotliv et al., 2005). Although the number of inorganic materials shown to be natural targets for protein binding is growing, proteins known to recognize periodic inorganic structures are still rare. We hypothesize that other surface-binding functionalities are encoded in protein sequence space and that non-natural proteins able to recognize and distinguish repetitive 2D targets can be identified by design or selection. Along these lines, others have selected by phage display and related techniques peptides with purported ability to bind and distinguish non-biological surfaces (Brown, 1992; Whaley et al., 2000). The drawback to a random peptide-based approach is that binding may occur via general association of an ensemble of peptide conformations with the surface rather than due to selection of a particular structure. This conclusion is based on the predominance of certain functional groups but the lack of a true consensus sequence for peptides selected on, for example, semiconductor surfaces (Whaley et al., 2000). We sought instead to take advantage of the fact that the b-helical THP domain is pre-evolved for recognition of a crystalline lattice. We chose for our study the Tenebrio molitor THP (TmTHP), a compact, right-handed b-helix composed of 12-amino acid repeats that form tight, internally disulfide bonded loops (Liou et al., 2000b) (Fig. 1). A Thr-Cys-Thr sequence from each repeat forms the strands of a parallel b-sheet spanning the length and width of the protein. The cysteines participate in disulfide bonds in the protein core, whereas the threonine side chains project outwards in an orderly array. Disulfide bridging and interstrand hydrogen bonding make the TmTHP protein particularly rigid (Daley et al., 2002; Daley and Sykes, 2004). We probed the tolerance of the TmTHP fold to multiple amino acid substitutions on the ice-binding face. Previously, limited site-directed mutagenesis was done to study the resilience of antifreeze activity to mutation (Marshall et al., 2002). In our study, we explore the sequence space of the disulfide-bonded b-helical fold of TmTHP regardless of its function as an antifreeze protein, with the expectation that specificity for other surfaces may replace ice-binding activity in certain mutants. We also address the contribution of the threonine grid to the architecture of this repeat protein. Our findings demonstrate that the TmTHP fold tolerates extens (...truncated)


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Maya Bar, Tali Scherf, Deborah Fass. Two-dimensional surface display of functional groups on a β-helical antifreeze protein scaffold, Protein Engineering Design and Selection, 2008, pp. 107-114, 21/2, DOI: 10.1093/protein/gzm070