Two-dimensional surface display of functional groups on a β-helical antifreeze protein scaffold
Maya Bar
2
Tali Scherf
1
Deborah Fass
2
0
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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.
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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)