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'