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tel-00605443, version 1 - 1 Jul 2011.
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tel-00605443, version 1 - 1 Jul 2011.
`a mes parents et `a C´eline.
tel-00605443, version 1 - 1 Jul 2011derriere dedicaces
tel-00605443, version 1 - 1 Jul 2011Acknowledgements :
I would like first to thank my direct team, Andy Fitch, Ir`ene Margiolaki
and Jon Wright for all the inestimable help, all the advice and ideas during
the last three years.
I would like to thank the three students from the University of Bath who
worked hard with me, Sotonye Dagogo, Ines Collings and Lucy Saunders.
The ID31, ID11, ID14 and BM16 teams for their support and help during
experiments.
John Helliwell, Serge P´erez and Marc Schiltz for accepting to be part of the
jury of this thesis.
Marion Giffard, Richard Kahn, Fran¸coise Bonnet´e, Mohamed El-Hajji and
Sanofi-Aventis for their collaboration on the polymorphism of urate oxidase.
Mathias Norrman, Gerd Schluckebier and Novo Nordisk for the insulin sam-
ples.
Elaine Chiu and Peter Metcalf for their collaboration on the baculovirus
project.
NicolasPapageorgiou, BrunoCoutard, ViolaineLantezandErnestGouldfor
their collaboration on the macro domain of the Mayaro virus project.
Finally, I want to thank the ESRF for financial support and the provision of
synchrotron beam time.
tel-00605443, version 1 - 1 Jul 2011derriere remerciements
tel-00605443, version 1 - 1 Jul 20111
Abstract:
Knowledgeofthestructureofproteinshelpsinunderstanding their biolog-
ical function. The main technique used, single crystal diffraction, requires the
limiting step of growing a single crystal. On the other hand, powder diffrac-
tion requires only a crystalline precipitate made of many microcrystals such
as those often discarded during the search for suitable single crystal growth
conditions. We present in this thesis different studies of proteins by powder
diffraction. Development of new methods for sample preparation and data
acquisition are also presented, as they have been crucial steps to obtain high
quality diffraction data.
We studied the polymorphism of Urate oxidase by observing the differ-
ent crystallographic phases resulting from the changes in the crystallisation
conditions. Acrystallographicphaseofpharmaceuticalinteresthasbeeniden-
tified.
Also one phase of urate oxidase complexed with its inhibitor gave a powder
pattern sufficient to re-determine and refine its structure.
A protocol for cryocooling protein powder samples has been found, extending
the lifespan of the sample in the intense X-ray beam. This allowed the refine-
ment from powder data of two forms of cryocooled human insulin.
We present also the determination of a preliminary structure of the mayaro
virus macro domain, based on a powder diffraction pattern obtained on a
single urchin-like bundle of needles.
A study of the protective protein matrix of two baculoviruses is presented,
showing some current limits of the method.
In annexes, the steps for preparing and analysing protein powders are de-
scribed.
tel-00605443, version 1 - 1 Jul 2011derriere abstract
tel-00605443, version 1 - 1 Jul 20113
R´esum´e de la th`ese :
Latechniquedediffractiondemonocristauxestlaplusutilis´eepourd´eterminer
unestructuretridimensionnelledeprot´eine,permettantainsilacompr´ehension
de sa fonction biologique. Cependant, cette technique n´ecessite l’obtention
d’un monocristal. La d´etermination d’une structure par diffraction de poudre
ne requiert que l’obtention d’un pr´ecipit´e cristallin, souvent obtenu lors de la
recherche d’une condition de cristallisation.
Nous pr´esentons dans cette th`ese plusieurs prot´eines ´etudi´ees par diffrac-
tion de poudre. Le d´eveloppement de nouvelles m´ethodes pour la pr´eparation
des ´echantillons et l’acquisition des donn´ees sont pr´esent´ees, ´etant donn´ee
leur importance cruciale dans ces ´etudes.
Nousavons´etudi´elepolymorphismedel’urateoxidaseparlad´etermination
des diff´erentes phases cristallines r´esultant des modifications des conditions
de cristallisation. Cette ´etude a` d´ebouch´e sur l’identification d’une forme
cristalline nouvelle d’int´erˆet pharmaceutique.
Une des phases d’urate oxidase a` permis l’obtention d’un clich´e de diffraction
de qualit´e suffisante a` la red´etermination et l’affinement de sa structure.
Un protocole pour le refroidissement cryog´enique de poudre de prot´eine est
pr´esent´e, offrant une dur´ee de vie accrue de l’´echantillon lors de l’acquisition
de donn´ees. Ce protocole a permis l’affinement de deux structures d’insuline
humaine.
Nous pr´esentons ´egalement la d´etermination d’une structure pr´eliminaire du
domaine macro du virus Mayaro, bas´e uniquement sur des donn´ees acquises
sur un unique ´echantillon polycristallin en forme d’oursin. Une ´etude de la
matrice de protection de deux baculovirus illustre les limites auxquelles se
heurte actuellement la technique de diffraction de poudre de prot´eines.
En annexes, les ´etapes n´ecessaires pour la pr´eparation et l’analyse des
donn´ees sont pr´esent´ees.
tel-00605443, version 1 - 1 Jul 2011derriere resume
tel-00605443, version 1 - 1 Jul 2011