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<strong>EMBL</strong> <strong>Grenoble</strong><br />
Structural biology of eukaryotic complexes in<br />
gene expression<br />
Previous and current research<br />
An intense focus of current biological research efforts is the elucidation of protein interaction networks<br />
(interactome). Many large multiprotein complexes are discovered. This poses considerable<br />
challenges for molecular level studies, in particular for eukaryotic multiprotein complexes with intracellular<br />
quantities refractory to large-scale extraction from source. Our research is focussed on<br />
developing new technologies to obtain, within a reasonable time-frame, well-defined and homogeneous<br />
samples of human multiprotein assemblies in transcription and hereditary disease, which<br />
we then use for high-resolution structural and functional analyses.<br />
Our major methodologies include molecular biology and X-ray crystallography. However, we readily<br />
apply techniques from other fields that are required for our research, both by ourselves and<br />
through collaborative efforts. A prerequisite for successful structural study of many complexes,<br />
both by electron microscopy and X-ray crystallography, is production of homogeneous, stable<br />
specimens. Present recombinant expression methods often require considerable investment in<br />
both labour and materials before multiprotein expression, and after expression and biochemical<br />
analysis do not provide flexibility for expressing an altered multiprotein complex. To meet these<br />
demands, we have introduced MultiBac, a modular, baculovirus-based system specifically designed<br />
for eukaryotic multiprotein expression (Berger et al., 2004, Nat. Biotechnol.). Recently, we<br />
have harnessed homologous and site-specific recombination methods in tandem for all steps involved<br />
in multigene assembly, thus providing a flexible, automatable platform for generation of multiprotein expression vectors and their<br />
rapid regeneration for revised expression studies. We have successfully implemented all steps involved in a robotics setup. By using our technology,<br />
we produced numerous large multiprotein assemblies in sufficient quantity and quality for structural studies, including the presumed<br />
~700 kDa heterodecamer scaffold of human TFIID general transcription factor.<br />
Future projects and goals<br />
At <strong>EMBL</strong> <strong>Grenoble</strong>, we will continue to advance our expression technologies<br />
to entirely automate and standardise the process of production<br />
for eukaryotic gene regulatory multiprotein complexes including<br />
the entire human TFIID holoenzyme and its various isoforms. In collaboration<br />
with the Schaffitzel group (page 96), we will subject the<br />
complex specimens produced to electron microscopic analyses. We<br />
will use homogeneous complexes thus identified for X-ray crystallography.<br />
By enlisting state-of-the-art mass spectrometric methods from systems<br />
biology, we will address a further bottleneck in complex crystallography,<br />
namely the challenge of defining crystallisable core<br />
assemblies of multiprotein complexes in a reasonable time frame (in<br />
collaboration with ETH Zürich). Also, we will expand our multiprotein<br />
expression strategies to prokaryotic and mammalian hosts.<br />
Imre Berger<br />
PhD 1995, MIT Cambridge<br />
and Leibniz University,<br />
Hannover.<br />
Postdoctoral research at MIT<br />
and the Institute of Molecular<br />
Biology and Biophysics<br />
(IMB), ETH Zürich.<br />
Habilitation 2005, ETH.<br />
Group leader at IMB from<br />
2005.<br />
Group leader at <strong>EMBL</strong> since<br />
2007.<br />
We develop and utilise advanced, automated technologies to produce<br />
eukaryotic multiprotein complexes for structural and functional<br />
analysis by a variety of methods including X-ray crystallography.<br />
Selected references<br />
Bieniossek, C. & Berger, I. (2009). Towards eukaryotic structural<br />
complexomics. J. Struct. Funct. Genomics, 10, 37-6.<br />
Bieniossek, C., Richmond, T.J. & Berger, I. (2008). MultiBac:<br />
multigene baculovirus-based eukaryotic protein complex production.<br />
Curr Protoc Protein Sci., Chapter 5, Unit 5.20<br />
Fitzgerald, D.J., Berger, P., Schaffitzel, C., Yamada, K., Richmond,<br />
T.J. & Berger, I. (2006). Protein complex expression by using<br />
multigene baculoviral vectors. Nature Methods, 3, 1021-1032.<br />
Schaffitzel, C., Oswald, M., Berger, I., Abrahams, J.P., Koerten, H.,<br />
Koening, R. & Ban, N. (2006). Structure of the E. coli signal<br />
recognition particle bound to a translating ribosome. Nature, ,<br />
503-506.<br />
89
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