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Institut de Biologie StructuraleGrenoble / France

Hart team

Team Leader: Darren Hart


Presentation

The Hart team uses combinatorial molecular biology methods to study enzymes of biological and medical interest.
Combinatorial methods (e.g. directed evolution, phage display) address problems that are too complex for rational design approaches. Mimicking Darwinian evolution in a test tube, large random libraries of variants are screened to identify rare hits with the desired property. In our ESPRIT process, for example, all truncations of a target protein are generated and screened using advanced picking and arraying robotics. Consequently, we are able to study certain biological questions with advantages over classical approaches.

Figure 1 : Screening tens of thousands of expression constructs of a target gene. Constructs are made as a random library and printed on membranes for soluble expression analysis by hybridisation of fluorescent antibodies.

Members

Darren Hart (Research Director CNRS) Google Scholar profile
Philippe Mas (Engineer CNRS)
Erasmus visiting scientist
Ph.D. student - recruiting
Post-doctoral researcher - recruiting

Research topics

Phage display libraries: We are combining well-established phage display protocols for antibody and peptide evolution with high-throughput robotics. Projects on nanobody selection and viral polymerase inhibition are in progress. Collaboration: Steyaert Lab, VRM group
Influenza host adaptation: Currently circulating avian influenza viruses may cross the species barrier and become highly pathogenic, human transmissible strains with pandemic potential. This can result from mutations in several influenza proteins. We are now characterising the interactions of these mutants with host cell factors using both structural and biophysical methods with the aim of understanding mechanisms of influenza host adaptation. Collaborations: Blackledge and Cusack groups

Figure 2 : A previously unsuspected domain from influenza polymerase, identified by HT expression screening of tens of thousands of random DNA constructs and structurally characterised by X-ray crystallography. A single mutation to lysine at residue 627 (A) can be responsible for the evolution of human influenza viruses from wild-type avian viruses that have a glutamic acid at this position (B). The mutation of residue 627 reinforces or disrupts a striking basic surface patch and we are seeking to understand how this affects polymerase function.

Histone deacetylases (HDACs):
These enzymes modify chromatin structure and regulate gene expression. Malfunctions result in disease including several cancers. We engineered well-expressing, catalytically active constructs of a HDAC and its partners. This allows us to study the enzyme and its interactions using X-ray crystallography, NMR and enzymatic assays. Collaboration: Blackledge group

Keywords

Directed evolution, protein engineering, host-pathogen interactions, viral polymerases

Specialized techniques

Automated clone screening, phage display

Available services

ESPRIT random library screening to identify soluble variants of challenging proteins. Access and funding available via Instruct

Major publications

Google Scholar profile for comprehensive list

1. Yerabham ASK, Mas PJ, Decker C, Soares DC, Weiergräber OH, Nagel-Steger L, Willbold D, Hart DJ, Bradshaw NJ, Korth C (2017) A Structural Organization for Disrupted in Schizophrenia 1, Identified by High-Throughput Screening, Reveals Distinctly Folded Regions Which Are Bisected by Mental Illness-Related Mutations. J. Biol. Chem.:jbc.M116.773903.
2. Leupold S, Büsing P, Mas PJ, Hart DJ, Scrima A (2017) Structural insights into the architecture of the Shigella flexneri virulence factor IcsA/VirG and motifs involved in polar distribution and secretion. J. Struct. Biol. Available at: http://www.sciencedirect.com/science/article/pii/S1047847717300370 [Accessed March 20, 2017].
3. Thierry E, Guilligay D, Kosinski J, Bock T, Gaudon S, Round A, Pflug A, Hengrung N, El Omari K, Baudin F, Hart DJ, Beck M, Cusack S (2016) Influenza Polymerase Can Adopt an Alternative Configuration Involving a Radical Repacking of PB2 Domains. Mol. Cell 61:125–137.
4. Pumroy RA, Ke S, Hart DJ, Zachariae U, Cingolani G (2015) Molecular Determinants for Nuclear Import of Influenza A PB2 by Importin α Isoforms 3 and 7. Structure 23:374–384.
5. Delaforge E, Milles S, Bouvignies G, Bouvier D, Boivin S, Salvi N, Maurin D, Martel A, Round A, Lemke EA, Ringkjøbing Jensen M, Hart DJ, Blackledge M (2015) Large-Scale Conformational Dynamics Control H5N1 Influenza Polymerase PB2 Binding to Importin α. J. Am. Chem. Soc. 137:15122–15134.
6. Sukackaite R, Jensen MR, Mas PJ, Blackledge M, Buonomo SB, Hart DJ (2014) Structural and Biophysical Characterization of Murine Rif1 C Terminus Reveals High Specificity for DNA Cruciform Structures. J. Biol. Chem. 289:13903–13911.
7. Reich S, Guilligay D, Pflug A, Malet H, Berger I, Crépin T, Hart D, Lunardi T, Nanao M, Ruigrok RWH, Cusack S (2014) Structural insight into cap-snatching and RNA synthesis by influenza polymerase. Nature 516:361–366.
8. Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr. Opin. Struct. Biol. 23:403–408.
9. Boivin S, Hart DJ (2011) Interaction of the influenza A virus polymerase PB2 C-terminal region with importin alpha isoforms provides insights into host adaptation and polymerase assembly. J. Biol. Chem. 286:10439–10448.
10. Yumerefendi H, Tarendeau F, Mas PJ, Hart DJ (2010) ESPRIT: an automated, library-based method for mapping and soluble expression of protein domains from challenging targets. J. Struct. Biol. 172:66–74.