RESEARCH
Nanotechnology of Polyproline
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Small molecules has been wide used to manipulate biological activities through protein receptors, and this strategy has become the norm in the development of inhibitor and probes. While protein arrangement at higher order also control or regulate biological processes such as recognition and signaling, it is difficult to manipulate such processes by molecules provided by general synthetic approach because the scale of proteins is above nanometers. To precise control the locations of small molecules to manipulate protein functions, we exploit synthetic biomolecules as scaffolds.
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We construct nanoscaffolds with polyproline peptide as these biomolecules form well-defined polyproline helix II (PPII) structure that has three fold symmetry and 0.9 nm pitch for every three proline residues. By installing chemical handle into proline building block for SPPS and controlling the peptide sequence, small molecule (ligand) location, distance and direction can be controlled at the range of few nanometers.
Project 1: Polyproline-based microarray and inhibitors
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Oligomeric protein-ligand interactions play essential roles in recognition and have become targets for inhibitors development. Designing multivalent inhibitors would require information of protein oligomer binding site distance, which usually rely on tedious protein structural studies. A microarray system that controls ligand distance on the surface can analyze the spatial requirement for protein oligomer and rapidly provide information for inhibitor design.
Fluorous anchors were installed to the same direction of the poyproline helix and allow immobilization of peptide to fluorous surface. The ligands were conjugation to another direction of the helix with controlled distance. In two protein models, the microarray system successfully provide estimation for the binding site distance. Based on the information, potent divalent inhibitor can be developed.
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In one example, polyproline was used as scaffold for inhibitor to LecA, a key lectin in P. aruginosa infection and resistance. Different galactose derivatives were tested to find a potent inhibitor in a rapid fashion. We are exploiting this microarray system on other multivalent protein targets.
Project 2: Controlled Ligand pattern on polyproline helix macrocycle
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Many protein receptors appear in oligomers and this is common for lectins. For example, some human lectins use this multivalent capability to recognize pathogens through glycan ligand density. To control ligand presentation in a universal approach, we connect several polyproline helix into a macrocycle. The assembly on polyproline helices can be efficiently carried out on a solid support with CuAAC reaction and further cyclized into macrocycles. As the ligand position on each helix can be controlled by SPPS and the helix assembly is stepwise, the individual ligands can be controlled into asymmetrical pattern.
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Using lectin model HPA, we observed the selectivity can arise from the ligand pattern on polyproline helix macrocycle scaffold. This system was further tested to label ASGPR receptor on Hep3B cell surface and suggested as a tool to probe receptor arrangement on live cells.
The macrocycle scaffold was extended from tri-helix to tetra-helix to expand different ligand patterns. In glycoscience, glycan cross-reactivity is usually a complicated issue that inhibitors commonly gave poor selectivity. By controlling ligand patterns, DC-SIGN (tetramer) selectivity can be boosted over langerin (trimer). This strategy provide a novel approach to deal with the glycan selectivity problem.
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Project 3: Polyproline conformation
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Our applications of polyproline peptides as molecular scaffolds are based on defined polyproline helix II structure. In fact the polyproline has two favorable conformation polyproline helix I and II. The helix II (PP II) is preferred in more polar aqueous solution, while helix I (PP I) can be observed in less polar environment such as n-propanol. The further understand the impact of the chemical modification on the change of the helix conformation, we introduce artificial tether to constrain the peptides. Such peptide stapling was performed in a systematic manner to show that both tether length and location affect the outcome of polyproline conformation.
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