Aggregate structure of hydroxyproline-rich glycoprotein (HRGP) and HRGP assisted dispersion of carbon nanotubes
Ben Wegenhart
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Li Tan
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Michael Held
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Marcia Kieliszewski
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Liwei Chen
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B. Wegenhart L. Tan M. Held M. Kieliszewski L. Chen (&) Department of Chemistry and Biochemistry, Ohio University
,
Athens, Ohio 45701, USA
Hydroxyproline-rich glycoproteins (HRGP) comprise a super-family of extracellular structural glycoproteins whose precise roles in plant cell wall assembly and functioning remain to be elucidated. However, their extended structure and repetitive block co-polymer character of HRGPs may mediate their self-assembly as wall scaffolds by like-with-like alignment of their hydrophobic peptide and hydrophilic glycopeptide modules. Intermolecular crosslinking further stabilizes the scaffold. Thus the design of HRGP-based scaffolds may have practical applications in bionanotechnology and medicine. As a first step, we have used single-molecule or single-aggregate atomic force microscopy (AFM) to visualize the structure of YK20, an amphiphilic HRGP comprised entirely of 20 tandem repeats of: Ser-Hyp4-Ser-Hyp-Ser-Hyp4-TyrTyr-Tyr-Lys. YK20 formed tightly aggregated coils at low ionic strength, but networks of entangled chains with a porosity of ~0.5-3 lm at higher ionic strength. As a second step we have begun to design HRGPcarbon nanotube composites. Single-walled carbon nanotubes (SWNTs) can be considered as seamless cylinders rolled up from graphene sheets. These unique all-carbon structures have extraordinary aromatic and hydrophobic properties and form aggregated bundles due to strong inter-tube van der Waals interactions. Sonicating aggregated SWNT bundles with aqueous YK20 solubilized them presumably by interaction with the repetitive, hydrophobic, Tyr-rich peptide modules of YK20 with retention of the extended polyproline-II character. This may allow YK20 to form extended structures that could potentially be used as scaffolds for site-directed assembly of nanomaterials.
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Published online: 1 August 2006
to the authors 2006
Hydroxyproline-rich glycoproteins (HRGPs) comprise
a superfamily of extra-cellular structural proteins
expressed in plant cell walls and extracellular matrix
during normal development and in response to stress
[1, 2]. HRGPs are extended macromolecules consisting
of small repetitive peptide and glycopeptide motifs.
While the peptide motifs often contain hydrophobic
tyrosine residues, the glycopeptide motifs result from a
combination of post-translational modifications unique
to plants, namely proline hydroxylation and
subsequent hydroxyproline (Hyp) glycosylation. The
precise oligosaccharides or polysaccharide decoration
pattern is driven by a sequence-dependent
glycosylation code [24]. The key to this glycosylation code is
Hyp contiguity: contiguous Hyp residues direct the
addition of small arabinooligosaccharides to Hyp,
while clustered non-contiguous Hyp residues direct the
addition of larger complex hetero-polysaccharides. The
addition of short oligosaccharides to Hyp residues
locks the contiguous Hyp-rich glycopeptide motifs into
an extended, left-handed polyproline-II helix
conformation and thus results in rigid hydrophilic regions.
In contrast, regions that lack contiguous Hyp
remain flexible while subsequent addition of long
polysaccharide to clustered non-contiguous Hyp
residues promotes an extended random coil conformation
[3].
Some HRGPs also contain hydrophobic,
tyrosinerich peptide motifs that function in intra- and
intermolecular crosslinking. Indeed, using a synthetic gene
approach we recently expressed in tobacco cells a
simple arabinosylated HRGP analog containing 20
tandem repeats of the sequence:
Ser-Hyp4-Ser-HypSer-Hyp4-Tyr-Tyr-Tyr-Lys, designated YK20, and
demonstrated that YK20 was extensively crosslinked
enzymically in vitro to give tyrosine-based
intermolecular crosslinks [5]. This indicated that YK20 rapidly
aligns itself for subsequent intermolecular crosslinking
and raised questions about the aggregate structure of
YK20 that drives this self-assembly, the networks that
arise and whether or not their properties can be
tailored for specific applications.
Here we report the first visualization of an YK20
network by the single-molecule or single-aggregate
imaging approach using atomic force microscopy
(AFM), the first such characterization for any HRGP.
We also noted that YK20, an amphiphilic molecule,
interacted with single-walled carbon nanotubes
(SWNTs) and dispersed SWNTs in aqueous solutions,
which raised the possibility that SWNT-YK20
complexes might be exploited to yield templates for the
assembly of high order structures.
YK20 synthetic gene construction, plant cell
transformation and YK20 glycoprotein isolation
A synthetic gene, YK20-EGFP, encoding 20 tandem
repeats of the protein sequence
Ser-Pro4-Ser-Pro-SerPro4-Tyr-Tyr-Tyr-Lys fused to the gene for the
enhanced green fluorescent protein (EGFP; Clontech)
was constructed, tobacco cells (Bright Yellow 2)
transformed, and the YK20 glycoprotein isolated after
EGFP removal, all as previously (...truncated)