Presentation Title

**From Sequence to Activity: Synthetic Neuroexcitatory Peptides from Fish-Hunting Cone Snails** Exemplary Presentation

Presenter Information

Mark Gad, Occidental CollegeFollow

Faculty Mentor

Dr. Joseph Schulz

Start Date

18-11-2017 2:15 PM

End Date

18-11-2017 2:30 PM

Location

9-255

Session

Bio Sciences 1

Type of Presentation

Oral Talk

Subject Area

biological_agricultural_sciences

Abstract

Highly selective natural toxins obtained from venomous animals are useful as research reagents and therapeutics. The potency of venom from fish-hunting cone snails on vertebrates and their unique prey-capture mechanism has made them excellent candidates for venom peptide studies. Within the venom of some fish-hunters, like Conus catus, we find the A-superfamily of peptides, which contains a family of neuroexcitatory peptides known to cause tetanic paralysis due to overstimulation of neurons. The precise molecular mechanism by which neuroexcitatory peptides cause tetanic paralysis and the identity of the molecular targets are currently under investigation. Our main goal was to generate fully folded and disulfide bonded forms of the two neuroexcitatory peptides, c4g and c4r. First, we obtained a crude version of the synthesized peptide then purified it using Reverse Phase-High Pressure Liquid Chromatography (HPLC). Secondly, we used a glutathione buffering system to transform the purified peptides into an active, fully folded conformation of the peptides. Three disulfide bonds characterize the fully folded conformation. Two ways were then used to test the presence of disulfide bonds in our peptides. RP-HPLC confirmed that after applying our oxidation conditions that we formed a new conformation of the peptides. Afterwards, we used mass spectrometry to confirm that the peptides with the new conformation have lost six daltons each. The loss of six daltons confirms that the process of synthesizing fully folded peptides has been successful. We next plan on determining the specific acitivity of these peptides using the spinal motility assay developed in our lab. We expect them to be highly active. We also plan on testing our methodology on other neuroexcitatory peptides that encode similar sequences as c4g and c4r.

Summary of research results to be presented

I will present chromatograms from running crude peptide samples on Reverse Phase High-Pressure Liquid Chromatography (RP-HPLC). Crude peptide chromatograms have many peaks which represent many impurities within the sample. I will compare this chromatogram from crude peptide to that of a pure peptide. I am able to purify the peptide after many runs on the RP-HPLC. I collect the main peaks of these run and refractionate them until the sample is pure. This pure sample is then ready for folding. I will also present the results of how a glutathione buffering system changed the conformation of the peptide using an RP-HPLC chromatogram. This chromatogram will demonstrate how a new conformation of the peptide was obtained after the application of the buffering system. I will then finally also present a mass spectrometry spectrum that shows a loss of six daltons and a formation of disulfide bonds. The spectra prove that the change in conformation detected by the HPLC is indeed an oxidation of the peptides

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Nov 18th, 2:15 PM Nov 18th, 2:30 PM

**From Sequence to Activity: Synthetic Neuroexcitatory Peptides from Fish-Hunting Cone Snails** Exemplary Presentation

9-255

Highly selective natural toxins obtained from venomous animals are useful as research reagents and therapeutics. The potency of venom from fish-hunting cone snails on vertebrates and their unique prey-capture mechanism has made them excellent candidates for venom peptide studies. Within the venom of some fish-hunters, like Conus catus, we find the A-superfamily of peptides, which contains a family of neuroexcitatory peptides known to cause tetanic paralysis due to overstimulation of neurons. The precise molecular mechanism by which neuroexcitatory peptides cause tetanic paralysis and the identity of the molecular targets are currently under investigation. Our main goal was to generate fully folded and disulfide bonded forms of the two neuroexcitatory peptides, c4g and c4r. First, we obtained a crude version of the synthesized peptide then purified it using Reverse Phase-High Pressure Liquid Chromatography (HPLC). Secondly, we used a glutathione buffering system to transform the purified peptides into an active, fully folded conformation of the peptides. Three disulfide bonds characterize the fully folded conformation. Two ways were then used to test the presence of disulfide bonds in our peptides. RP-HPLC confirmed that after applying our oxidation conditions that we formed a new conformation of the peptides. Afterwards, we used mass spectrometry to confirm that the peptides with the new conformation have lost six daltons each. The loss of six daltons confirms that the process of synthesizing fully folded peptides has been successful. We next plan on determining the specific acitivity of these peptides using the spinal motility assay developed in our lab. We expect them to be highly active. We also plan on testing our methodology on other neuroexcitatory peptides that encode similar sequences as c4g and c4r.