Abstract
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-Tyr-Tyr-Tyr-Lys. YK20 formed tightly aggregated coils at low ionic strength, but networks of entangled chains with a porosity of ~0.5–3 μm at higher ionic strength. As a second step we have begun to design HRGP-carbon 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.
Highlights
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]
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 described [5]
We have demonstrated that YK20, a genetically engineered HRGP, forms closely aggregated coils in low ionic strength solutions, and random networks of entangled chains at high ionic strength conditions
Summary
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]. 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 [2,3,4]. 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 results in rigid hydrophilic regions. Regions that lack contiguous Hyp remain flexible while subsequent addition of long
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