Structural and functional analysis of Cysteine loop receptors, Chorismatases and a C-type like Lectin protein

Abstract

The work presented here was focused on expression, purification, functional characterization and X-ray structure determination of proteins from three different protein families - Cysteine Loopreceptors, Chorismatases and C-type like lectin proteins.Cysteine loop receptors are ligand gated ion channels and are found in the invertebrate and the mammalian nervous system such as nicotinic acetylcholine receptors (nAChRs) and γ-aminobutyric acid receptors (GABAA). As a part of doctoral thesis, a new member of Cysteine Loopreceptors was discovered from a thermophilic worm Alvinella pompejana and was named Alv-a1-pHCl. The Alv-a1-pHCl receptor was functionally characterized in Xenopus oocytes. Further, Alv-a1-pHCl protein was expressed in Sf9 cells, purified and assayed for thermostability andcrystallization attempts were made.An alpha-bungarotoxin affinity based purification method was developed and used for purification of nAChR from Torpedo californica. The stability of alpha-bungarotoxin-nAChR complex was determined in different detergents and crystallization attempts were made. Furthermore attempts were made to develop an expression system for the alpha 7 nAChR from Rattus norvegicus in Drosophila eyes, which was of no avail. The GABAA β3 receptor, an inhibitory neurotransmitter receptor from Rattus norvegicus was expressed in Sf9 cells, purified and assayed for stability in different detergents and crystallization was tried in different detergents in presence of ligands and lipids.Chorismatases are enzymes involved in chorismate metabolism. As a recently discovered family ofenzymes, no structural and functional details were available at the outset of this doctoral work.Two different homologues, FkbO and Hyg5 were expressed in E.coli, purified and crystallized at a resolution of 1 Å and 1.9 Å in a complex with the competitive inhibitor3-(2-carboxyethyl)benzoate and a functional mechanism was proposed.Proteins belonging to the C-type lectin family are involved in sugar binding and cell-cellinteractions. Proteins with a C-type lectin domain in sea urchins such as SM50 have shown to be important in biomineralization. As no structural details were available of such proteins from sea urchins, the CTL domain of SM50 protein from sea urchin Strongylocentrotus purpuratus was purified and crystallized. The crystals diffracted to a resolution of 2.8 Å.The work presented in the thesis is part of following publications.1. Puneet Juneja, Reinhold Horlacher, Daniel Bertrand, Ryoko Krause, Fabrice Marger,Wolfram WelteJ Biol Chem. 2014 May 23;289(21):15130-15140. Epub 2014 Apr 9.An internally modulated, thermostable, pH sensitive Cys-loop receptor from the hydrothermal vent worm Alvinella pompejana.2. Juneja et al, Manuscript in preparation.Stability of Alpha-Bungarotoxin affinity purified Torpedo nicotinic acetylcholine receptor in lipid based detergents.3. Puneet Juneja, Florian Hubrich, Kay Diederichs, Wolfram Welte, Jennifer N. AndexerJ Mol Biol. 2014 Jan 9;426(1):105-15. doi: 10.1016/j.jmb.2013.09.006Mechanistic implications for the chorismatase FkbO based on the crystal structure.4. Juneja et al, Manuscript in preparation.Crystal structure and mechanism of Hyg5 type chorismatase.5. Puneet Juneja, Ashit Rao, Helmut Cölfen, Kay Diederichs, Wolfram WelteActa Crystallogr F Struct Biol Commun. 2014 Feb 1;70 (Pt 2):260-2.Crystallization and preliminary X-ray analysis of the C-type lectin domain of the spicule Matrixprotein SM50 from Strongylocentrotus purpuratus.