http://www.esrf.eu/Jobs/Research/CFR355
Subject: Kinetic Crystallography to Probe for Catalytic Mechanism and
Protein Loop Motions in Glycosyltransferases
General Framework: Glycosyltransferases are an enormous class of enzymes
responsible for the biosynthesis of oligosaccharides, polysaccharides
and glycoconjugates. They catalyse the transfer of a sugar from a donor
substrate, usually a nucleotide sugar, to an acceptor.
Glycosyltransferase reactions can occur with either retention or
inversion of the anomeric configuration of the transferred sugar. While
the catalytic mechanisms of glycoside hydrolyses are well characterized,
many uncertainties remain concerning those of glycosyltransferases. The
proposed project aims at investigating the catalytic mechanisms of an
inverting glycosyltransferase and of a retaining one, by kinetic
crystallography with the use of caged compounds based on the
nucleotide-sugar substrate.
Description of the thesis work: The successful candidate will be in
charge of the production of recombinant glycosyltransferases. He/she
will set up the crystallization assays of the enzymes and of the
complexes with the caged compounds provided by our chemist
collaborators. If needed, site-directed mutagenesis will be performed in
order to alter the kinetic characteristic of the reaction for
facilitating the kinetic crystallography study. The student will
optimize by microspectrophotometry the conditions for efficient
photocleavage of the caged compounds, first in frozen protein solutions,
then in crystals. Using the Temperature Derivative Fluorescence
Microspectrophotometry method, he will find a temperature range in which
solvent rearrangements will allow the enzymatic reaction to proceed.
He/she will then collect diffraction data on protein crystals to solve:
(i) the structure of the protein/caged compound complex; (ii) the
structure of the protein/end product complex after complete cleavage by
strong light irradiation; (iii) the structure of putative intermediate
states of the enzymatic reaction. Overall, these data should provide
snapshots of the catalytic mechanism and protein loop motions.
Place of Work: ESRF in Grenoble.
Supervisors: Dr. Antoine Royant ((+33) (0)4 76 88 17 46; antoine.royant
at esrf.fr) & Dr. Serge Pérez (+33) (0)4 76 88 21 81; serge.perez at
esrf.fr).
General Conditions: You should hold a degree in either Physical
Chemistry, Chemistry, Biochemistry or Structural Biology allowing
enrolment for a PhD, such as an MSc, Master 2 de Recherche, Laurea or
equivalent. Contract of two years renewable (subject to satisfactory
progress) for one year. Gross salary around 2268 €/month. (The applicant
will be responsible for arranging his/her academic registration and for
paying the fees (if any)). The ESRF is an equal opportunity employer and
encourages applications from disabled persons.
If you are interested, please send us an e-mail (recruitment at esrf.fr)
with your address, and we will provide you with an application form. Or
print out an application form on the World Wide Web
http://www.esrf.fr/Jobs/Applying. In addition to the application form,
you should provide us with a detailed CV and the names of two referees.
Deadline: 15-02-2010
Contract type: Non-permanent contract (CDD)
Please send your application (form, covering letter and CV) to: peritore
at esrf.fr with Subject: 'PhD Thesis Student (f/m), Kinetic
Crystallography to Probe for Catalytic Mechanism and Protein Loop
Motions in Glycosyltransferases'
