Peptides

Four-Helix Bundle Peptides (Postdoc with Prof. Reza Ghadiri)

A series of cavity creating mutations were made to a four-helix bundle peptide with a view towards producing binding and catalytic sites. The mutant peptides were characterized in solution by CD spectroscopy and size exclusion chromatography, and in the solid state by X-ray crystallography.


	Despite cavity forming and hydrophilic mutations in the hydrophobic core of the bundle, typically peptides were found to retain their solid state structures but with disordering and fraying associated with the mutation. Loss of thermal stability caused by disruption of the well packed core was evident in the CD thermal melts. Helix fraying was also apparent in solution from reduced size exclusion elution volumes.



				Left - Crystal structure of a four-helix bundle peptide with a cavity forming alanine residue in the hydrophobic core. The structure is in space group P4₁32 and the asymmetric unit of 2 helices is shown. The bundle is completed by a 2-fold rotation

A synthesis of Fmoc-p-azidotetrafluorophenylalanine was developed to enable incorporation of a photoaffinity labelling group into peptides by automated solid phase peptide synthesis. The residue was prepared from achiral starting materials and resolved enzymatically. A test peptide was successfully prepared using the modified residue and standard automated Fmoc chemistry demonstrating its stability to the conditions required for cleavage of common protecting groups (Pbf, Trt, tBu and Boc).

Cyclic Peptide Sequencing (Postdoc with Prof. Reza Ghadiri)

Cationic amphipathic cyclic peptides with alternating D and L amino acids have shown promise as antibacterial agents and are active against pathogens which have become resistant to conventional antibiotics. The antibacterial activity and toxicity against mammalian cells are dependent on the peptide sequence, so we undertook library synthesis to optimize these properties.

To enable rapid identification of compounds from split and pool combinatorial peptide synthesis using mass spectrometry, an automated sequencing program was developed.

The software takes into account the residue choices in the combinatorial synthesis, and is aware of common fragmentations of the backbone and side chain of peptides. It was tested using a variety of cyclic hexa- and octa-peptides, and a small split-and-pool library of hexapeptides.

DNA Binding Peptides (Postdoc with Dr. Shankar Balasubramanian)

Quadruplex structures can form in guanine rich tracts of DNA and are stabilized by Hoogsteen hydrogen bonding and coordination of cations. Such DNA sequences are present in the telomeres of chromosomes and are elongated by the enzyme telomerase which is active in many cancer cells. Telomerase has been shown to be inhibited by formation of quadruplexes which makes quadruplex stabilization a possible means of disrupting cancer cell replication. Similar G-rich sequences occur at other points in the human genome such as the immunoglobulin switch region and in certain regulatory elements including the insulin linked polymorphic region. We are interested in specific targeting of potential quadruplexes in the promoters of certain oncogenes, with the goal of gene down regulation.


	Peptides containing heterocyclic amino acids and proteins based on modified zinc fingers were investigated for their ability to interact with G-quadruplexes.

We also prepared amino acids and capping groups capable of end stacking with guanine tetrads to enable combinatorial synthesis of unnatural peptides with a propensity for quadruplex binding. ELISA methodology was used to screen potential ligands from split and pool library synthesis, and mass spectrometry will be used for structure determination. It was found that the ELISA gave results comparable to established techniques such as surface plasmon resonance and FRET melting assays.


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