O Tethered Agonist At Three Selected Sites example essay topic

The tethered agonist approach to mapping ion channel proteins - toward a structural model for the agonist binding site of the acetylcholine receptor. Research by Linton g Li, We nge Zhong, Niki Zacharias, Caroline Gibbs, Henry A Lester, Dennis A. Dougherty. Condensation of the Research Purpose of the Study: The purpose of the study was to develop a structural model for the acetylcholine receptor and use newer techniques to more easily understand the structure and function of membrane proteins. Background: Understanding the integral membrane proteins of neurons and excitable cells is often difficult because they cannot be studied using tradition techniques of X-ray crystallography and NMR. Therefore to understand their function other techniques must be used to study them. The most useful approach that has been determined to analyze these proteins is to use two, site-directed mutagenesis and electrophysiology.

This approach has been improved by nonsense suppression methodology. With this a tethered agonist approach was used to map the agonist binding site of ligand-gated channels. This would provide invaluable information for understanding its response to pharmaceuticals and other chemicals in the human system. Previous studies indicated that sub-units near the binding site of the acetylcholine receptor may contribute to binding and may actually be the binding sites. Using the new method of tethering unnatural amino acids can provide information in determining where and how the binding site works. The use of unnatural amino acids allows researchers to incorporate them in multiple positions to pinpoint tether locations and eliminate proposed sites.

With new studies a better model for the agonist binding site of the acetylcholine receptor can be developed. Proteins with synthesized, unnatural amino acids were expressed on Xenopus oocytes to study the structure and function of the binding site. Researchers' Approach: 1. Synthesis of Tyr-OnQ and Tyr-O 3 Bin the unnatural amino acid technique the target is to the develop an "amino acid with the side chain in place, the amino group protected, and the carboxylate activated as a ester.

The usual method of forming the ester in the final step failed. It was found that the quaternary ammonium had to be added to the final step of the synthesis. The synthesis of Tyr-O 2 Bu was not complicated with other obstacles. 2. Incorporation of Tyr-OnQ into the nAChRThe unnatural amino acid Tyr-OnQ was added at multiple position of the receptor site.

For sites designated as 'a two copies of the synthesized side chain was used. For ~a and "a sites mutations were made to attach there. This was done so that both agonist binding sites were disturbed. With there unnatural amino acids mutant proteins were expressed on the Xenopus oocytes. Once expressed their whole cell currents were taken. The current is used to open the receptor.

It was established that large observed large currents were due to the acetylcholine receptor rather than the Xenopus oocyte. Only three of the evaluated sites produced significant enough current, indicating an active receptor, to be considered for further analysis. These sites were then treated with the natural amino acid of the acetylcholine receptor. This caused in increase in the observed current. This led to the conclusion that the quaternary ammonium group is a partial agonist. 3.

Incorporation of an isosteric, neutral tether. The Tyr-O 3 Bu chain was incorporation on the three proposed receptor sites. In this the quaternary ammonium is substituted with a term-butyl group. This chain does not produce currents as large or significant as the Tyr-OnQ group. It was deduced that with Tyr-O 3 Bu the sites were not activated because is it blocked by the term-butyl group. It was also found that less receptor sites were expressed on the oocytes.

Observations: o Tethered agonist at three selected sites gave active receptors. Variations of these agonist can discriminate between the different sites. o Nonsense suppression can be efficient if different unnatural amino acids are used. o Tyr-O 3 Bu does not activate other sites o Tyr-O 3 Q (another unnatural amino acid) activates sites. o For two of the receptor sites (of the three analyzed) the unnatural amino acids showed that they were less involved in the agonist binding site. This was shown by the disturbance the tether Tyr-O 3 Bu caused. o A tethered quaternary ammonium is more effective showing that the remote sites do play some role in binding. What Researchers Accomplished: The collection of information yielded by the tethered unnatural amino acids allowed a tentative model to be generated on the structure of the binding site. The results also yielded information on the function of specific sites on the acetylcholine receptor. Commentary on the Research: The research on using a tethered agonist approach to map the structure of the receptor is crucial for further knowledge in the field of biology, medicine and pharmaceuticals.

Being able to pinpoint as well as eliminate binding sites allows further insight in to field. Developing exact structures for these receptors is something that many researchers have been struggling to do. With new techniques to analyze the structure obtaining this information becomes easier. Summary: The technique of using a tethered agonist approach to analyze the structure and function of the acetylcholine receptor gave information on specific sites that participating in binding and yielded a provisional structure.