Deadline for full application: December 15th, 2013
Interviews: March, 2014
Start of the Ph.D.: October 1st, 2014
Department: Chemical and Structural Biology
Title of the PhD project: Structural characterization of allosteric states of a pentameric ligand-gated ion channel
Name of the lab: Structural Dynamics of Macromolecules
Head of the lab: Marc Delarue
PhD advisor: Marc Delarue
Email address: firstname.lastname@example.org
Web site address of the lab: http://lorentz.pasteur.fr
Doctoral school affiliation and University:
B3MI and Paris 7 University
Presentation of the laboratory and its research topics:
In the lab, we interested in adding an extra-dimension (time) to molecular and structural studies of biological macromolecules. Also, in all our projects we constantly try to associate an experimental approach with a computational one. Sometimes we have to develop our own methods.
The two biological systems we are interested in are i) DNA polymerases and ii) a pentameric ligand-gated ion channel, a bacterial homolog of the acetylcholine receptor. In the latter case, we seek a deep molecular understanding of the gating mechanism that would explain how the binding of the proper agonist in the extra-cellular domain can give rise to the opening of the pore in the transmembrane domain. Indeed, this would have a tremendous impact in the development of pharmacologically active molecules acting on this family of receptors, which also includes the glycine receptors, the 5HT3 receptors and the GABA receptors. In the case of polymerases, the goal is to understand in details all the steps of the chemical reaction performed by a polymerase in order to redesign its active site to make it accept different nucleotide substrates, leading to applications in synthetic biology. In all these cases we need to understand the dynamics of the protein, which can be approached by simulation methods (coarse-grained or with all atoms) but needs to be constrained by as many experimental constraints as possible, using for instance time-resolved crystallography.
Description of the project:
(Arial font size 11: 600 words in total with at least 50% dedicated specifically to the PhD project(s))
The nicotinic receptor has been studied for many years by Pr. J.-P. Changeux and his group in the Institut Pasteur and by a number of other labs throughout the world. It is a classic example of an allosteric protein: the binding of a ligand (neurotransmitter) in the extra-cellular domain elicits the opening of the pore in the trans-membrane domain, allowing a flow of ions through the membrane down their electrochemical gradient.
Members of the family include the acetylcholine receptors, the 5HT3 receptors (cationinc receptors), as well as the GABA and glycine receptors (anionic receptors). They are all essential in the propagation of the nervous signal through the synaptic cleft in between two adjacent nerve cells.
On the molecular level this system is a perfect one for studying the transduction of a signal at the molecular level either computationally or experimentally. We have an on-going collaboration with the group of P. J. Corringer in the NeuroSciences Department in the Pasteur Institute, to study the structure of a bacterial member of the Cys-Loop family, a proton-gated receptor from Gloeobacter violaceus called GLIC. Previously, we have solved the structure of GLIC in an open conformation, alone and in the presence of General Anaesthetics, as well as several mutants, alone or in the presence of Ethanol, shedding light on the process of activation or modulation of these receptors.
Recently, we also managed to solve the structure at neutral pH (pH 7), which displays all the expected features of the resting from: the channel is closed and the extra-cellular has undergone significant structural changes both at the tertiary and quaternary levels. However, the current resolution of these crystals is still relatively modest (4.5 Å).
To complete the structural description we need i) to get crystals diffracting at a better resolution for the neutral pH form (e.g. 3 Å) and ii) find a way to crystallize and solve the structure of the desensitized form.
Specifically, the PhD project would comprise the following sub-topics
-Learn to purify the protein and to crystallize it in both already known forms
-Get better diffracting and larger crystals of the acidic pH form, so as to perform neutron diffraction experiments in Grenoble ; this would allow to identify those acidic residues that are protonated at pH 4.5
-Get better diffraction and better reproductibility for neutral pH form in order to be able to routinely perform soaking experiments with e.g. general anaesthetics or other potential modulators. Among different things we plan to try to apply a high mechanical pressure on the crystals, try new controlled dehydration protocols at room temperature and to diffuse Xenon gas into the crystals.
-Get crystals of the desensitized form at acidic pH (one possibility is to avoid the use of classical detergents such as DDM and favor alternative ways to solubilize the protein such as nanopores)
-Enlarge the study to other members of the family, particularly chimeras constructs that combine eukaryotic and bacterial domains and that have been proved to be functional by the Corringer group.
This project would be particularly suited for a PhD student desiring to acquire hands-on experience on membrane protein crystallography and membrane protein structure determination on a member of a family with wide potential pharmacological interest.
Close collaborations with groups performing site-directed mutagenesis and functional studies using electrophysiology (P.J. Corringer’s lab, Institut Pasteur), or molecular dynamics simulations (M. Baaden’s lab, IBPC, Paris) studies are expected.
L. Sauguet et al., 2013, PNAS (submitted)
L. Sauguet et al., 2013, EMBO J. 32:728-41
L. Sauguet et al., 2013, Nature Comm. 4:1697
M. Prevost et al., 2012, Nature Struct. Mol. Biol. 19:642
H. Nury et al., 2011, Nature 69(7330):428-31
Crystallography, ligand-gated ion channel, allostery, structural biology, neuroscience
Expected profile of the candidate (optional):
-Strong background in molecular biology and biochemistry
-Strong interest in structural biology and crystallography
-Strong interest in molecular neurosciences as well as pharmacology
Contact: Marc Delarue, Institut Pasteur, Pasteur, (email@example.com)