Hart team : Research activities

i) The study and inhibition of viral replication using directed evolution methods ; ii) Expression of challenging proteins using high throughput random construct libraries.


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


FluPept : Novel Influenza Peptide Inhibitors by Directed Evolution (ANR-funded) : We aim to develop proof-of-concept and novel lead drug candidates that target viral and host protein complexes essential for influenza viral replication. These novel peptide-based molecules should show low levels of drug-resistance and will be applied to virus-infected epithelial cells of the upper respiratory tract using inhaler or spray-based technologies. Methods include phage display, biophysical assays, X-ray crystallography and cellular virus replication assays. Collaboration : Naffakh Lab, Institut Pasteur and I2BM.

Figure 1. A) Experimental approach to evolve high affinity cell-penetrating peptide or peptidomimetic inhibitors from low affinity natural peptide ligands (small linear motifs ; SLiMs) B) M13 phage display of SLiMs for affinity selections.

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. After identifying unpredicted soluble domains using ESPRIT (see below), we characterised their interactions with host cell factors involved in nuclear import and influenza virus host adaptation. Collaborations : Cingolani and Blackledge groups.

Figure 2 : A previously unsuspected domain from influenza polymerase, identified by ESPRIT expression screening and structurally characterised by X-ray crystallography. A single mutation to lysine at residue 627 can be responsible for the evolution of human influenza viruses from wild-type avian viruses that have a glutamic acid at this position ; this can be partly explained through the interactions between this region and the host factor ANP32.

Expression of Soluble Proteins by Random Incremental Truncation (ESPRIT)

ESPRIT random library screening to identify soluble variants of challenging proteins. For over a decade, we have used it on our targets and those of collaborators/users to identify well-expressing soluble constructs from poorly understood proteins.
Funded access to this unique technology is available via Instruct. Non- or partially funded access via FRISBI and ISBG.

Logo Hart team

Figure 3 : Libraries of 10-30,000 random constructs from a single target gene are generated in E. coli and screened using colony picking and arraying robotics. Clones that produce purifiable proteins are sequenced to determine domain boundaries.


See ORCID profile for complete details

1. Giachin G, Jessop M, Bouverot R, Acajjaoui S, Saïdi M, Chretien A, Bacia‐Verloop M, Signor L, Mas PJ, Favier A, Meneroud EB, Hons M, Hart DJ, Kandiah E, Erba EB, Buisson A, Leonard G, Gutsche I, Soler‐Lopez M (2021) Assembly of The Mitochondrial Complex I Assembly Complex Suggests a Regulatory Role for Deflavination. Angew. Chem. Int. Ed. 60:4689–4697.
2. Camacho-Zarco AR, Kalayil S, Maurin D, Salvi N, Delaforge E, Milles S, Jensen MR, Hart DJ, Cusack S, Blackledge M (2020) Molecular basis of host-adaptation interactions between influenza virus polymerase PB2 subunit and ANP32A. Nat. Commun. 11:3656.
3. Gao S, Jiang B, Zhu S, Hart DJ, Liu X, Wang H, An Y (2019) The K296-D320 region of recombinant levansucrase BA-SacB can affect the sensitivity of Escherichia coli host to sucrose. Ann. Microbiol. 69:1147-1154.
4. Wang H, Hart DJ, An Y (2019) Functional Metagenomic Technologies for the Discovery of Novel Enzymes for Biomass Degradation and Biofuel Production. BioEnergy Res. 12:457–470.
5. Simonini S, Mas PJ, Mas CMVS, Østergaard L, Hart DJ (2018) Auxin sensing is a property of an unstructured domain in the Auxin Response Factor ETTIN of Arabidopsis thaliana. Sci. Rep. 8:13563.
6. Liu X, Li T, Hart DJ, Gao S, Wang H, Gao H, Xu S, Zhang Y, Liu Y, An Y (2018) A universal mini-vector and an annealing of PCR products (APP)-based cloning strategy for convenient molecular biological manipulations. Biochem. Biophys. Res. Commun. 497:978–982.
7. Gao H, Qi X, Hart DJ, Gao S, Wang H, Xu S, Zhang Y, Liu X, Liu Y, An Y (2018) Three Novel Escherichia coli Vectors for Convenient and Efficient Molecular Biological Manipulations. J. Agric. Food Chem. 66:6123–6131.
8. Tarbouriech N, Ducournau C, Hutin S, Mas PJ, Man P, Forest E, Hart DJ, Peyrefitte CN, Burmeister WP, Iseni F (2017) The vaccinia virus DNA polymerase structure provides insights into the mode of processivity factor binding. Nat. Commun. 8:1455.
9. 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 the Disrupted in Schizophrenia 1 protein, identified by high-throughput screening, reveals distinctly folded regions, which are bisected by mental illness-related mutations. J. Biol. Chem. 292:6468–6477.
10. Sukackaite R, Cornacchia D, Jensen MR, Mas PJ, Blackledge M, Enervald E, Duan G, Auchynnikava T, Köhn M, Hart DJ (2017) Mouse Rif1 is a regulatory subunit of protein phosphatase 1 (PP1). Sci. Rep. 7:2119.
11. Mas PJ, Hart DJ (2017) ESPRIT : A Method for Defining Soluble Expression Constructs in Poorly Understood Gene Sequences. Methods Mol. Biol. 1586:45–63.
12. 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. 198:19–27.
13. Gao S, Yao S, Hart DJ, An Y (2017) Signal peptide-dependent protein translocation pathway is crucial for the sucrose sensitivity of SacB-expressing Escherichia coli. Biochem. Eng. J. 122:71–74.
14. Gao S, Qi X, Hart DJ, Gao H, An Y (2017) Expression and Characterization of Levansucrase from Clostridium acetobutylicum. J. Agric. Food Chem. 65:867–871.
15. Desravines DC, Serna Martin I, Schneider R, Mas PJ, Aleksandrova N, Jensen MR, Blackledge M, Hart DJ (2017) Structural Characterization of the SMRT Corepressor Interacting with Histone Deacetylase 7. Sci. Rep. 7:3678.
16. Simon B, Huart A-S, Temmerman K, Vahokoski J, Mertens HDT, Komadina D, Hoffmann J-E, Yumerefendi H, Svergun DI, Kursula P, Schultz C, McCarthy AA, Hart DJ, Wilmanns M (2016) Death-Associated Protein Kinase Activity Is Regulated by Coupled Calcium/Calmodulin Binding to Two Distinct Sites. Structure 24:851–861.
17. Pukáncsik M, Orbán Á, Nagy K, Matsuo K, Gekko K, Maurin D, Hart D, Kézsmárki I, Vertessy BG (2016) Secondary structure prediction of protein constructs using random incremental truncation and vacuum-ultraviolet CD spectroscopy. PloS One 11:e0156238.
18. Delaforge E, Milles S, Huang J-R, Bouvier D, Jensen MR, Sattler M, Hart DJ, Blackledge M (2016) Investigating the Role of Large-Scale Domain Dynamics in Protein-Protein Interactions. Front. Mol. Biosci. 3:54.
19. Yao S, Hart DJ, An Y (2016) Recent advances in universal TA cloning methods for use in function studies. Protein Eng. Des. Sel. 29:551–556.
20. 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.
21. 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.
22. Delaforge E, Milles S, Bouvignies G, Bouvier D, Boivin S, Salvi N, Maurin D, Martel A, Round A, Lemke EA, Jensen MR, 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.
23. 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.
24. 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.
25. Puranik S, Acajjaoui S, Conn S, Costa L, Conn V, Vial A, Marcellin R, Melzer R, Brown E, Hart D, Theißen G, Silva CS, Parcy F, Dumas R, Nanao M, Zubieta C (2014) Structural Basis for the Oligomerization of the MADS Domain Transcription Factor SEPALLATA3 in Arabidopsis. Plant Cell 26:3603–3615.
26. Hart DJ, Waldo GS (2013) Library methods for structural biology of challenging proteins and their complexes. Curr. Opin. Struct. Biol. 23:403–408.
27. Pumroy RA, Nardozzi JD, Hart DJ, Root MJ, Cingolani G (2012) Nucleoporin Nup50 Stabilizes Closed Conformation of Armadillo repeat 10 in Importin α5. J. Biol. Chem. 287:2022–2031.
28. Levdikov VM, Blagova EV, Rawlings AE, Jameson K, Tunaley J, Hart DJ, Barak I, Wilkinson AJ (2012) Structure of the Phosphatase Domain of the Cell Fate Determinant SpoIIE from Bacillus subtilis. J. Mol. Biol. 415:343–358.
29. Chatterjee J, Beullens M, Sukackaite R, Qian J, Lesage B, Hart DJ, Bollen M, Köhn M (2012) Development of a Peptide that Selectively Activates Protein Phosphatase-1 in Living Cells. Angew. Chem. Int. Ed Engl. 51:10054–10059.
30. Nadal M, Kaczmarska Z, Mas PJ, Blanco AG, Arnan C, Solà M, Hart DJ, Coll M (2011) Structural and functional characterization of human cytomegalovirus terminase leads to a new antiviral target. Antiviral Res. 90:A24.
31. Yumerefendi H, Desravines DC, Hart DJ (2011) Library-based methods for identification of soluble expression constructs. Methods San Diego Calif 55:38–43.
32. 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.
33. An Y, Yumerefendi H, Mas PJ, Chesneau A, Hart DJ (2011) ORF-selector ESPRIT : A second generation library screen for soluble protein expression employing precise open reading frame selection. J. Struct. Biol. 175:189–197.
34. An Y, Meresse P, Mas PJ, Hart DJ (2011) CoESPRIT : a library-based construct screening method for identification and expression of soluble protein complexes. PLoS One 6:e16261.
35. 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.
36. Ruigrok RW, Crepin T, Hart DJ, Cusack S (2010) Towards an atomic resolution understanding of the influenza virus replication machinery. Curr Opin Struct Biol 20:104–13.
37. Rawlings AE, Levdikov VM, Blagova E, Colledge VL, Mas PJ, Tunaley J, Vavrova L, Wilson KS, Barak I, Hart DJ, Wilkinson AJ (2010) Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen. Protein Eng. Des. Sel. PEDS 23:817–825.
38. Oger F, Lecorgne A, Sala E, Nardese V, Demay F, Chevance S, Desravines DC, Aleksandrova N, Le Guével R, Lorenzi S, Beccari AR, Barath P, Hart DJ, Bondon A, Carettoni D, Simonneaux G, Salbert G (2010) Biological and biophysical properties of the histone deacetylase inhibitor suberoylanilide hydroxamic acid are affected by the presence of short alkyl groups on the phenyl ring. J. Med. Chem. 53:1937–1950.
39. Nadal M, Mas PJ, Blanco AG, Arnan C, Solà M, Hart DJ, Coll M (2010) Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain. Proc. Natl. Acad. Sci. U. S. A. 107:16078–16083.
40. Chevanas S, Guilligay D, Cusack S, Hart DJ, Ruigrok WH, Crepin T (2010) Avancées dans l’étude structurale et la compréhension moléculaire du mécanisme de transcription du virus de la grippe. Virologie 14:435–45.
41. Boivin S, Cusack S, Ruigrok RWH, Hart DJ (2010) Influenza A virus polymerase : structural insights into replication and host adaptation mechanisms. J. Biol. Chem. 285:28411–28417.
42. Morinière J, Rousseaux S, Steuerwald U, Soler-López M, Curtet S, Vitte A-L, Govin J, Gaucher J, Sadoul K, Hart DJ, Krijgsveld J, Khochbin S, Müller CW, Petosa C (2009) Cooperative binding of two acetylation marks on a histone tail by a single bromodomain. Nature 461:664–668.
43. Dias A, Bouvier D, Crepin T, McCarthy AA, Hart DJ, Baudin F, Cusack S, Ruigrok RW (2009) The cap-snatching endonuclease of influenza virus polymerase resides in the PA subunit. Nature 458:914–8.
44. Bonneau F, Lenherr ED, Pena V, Hart DJ, Scheffzek K (2009) Solubility survey of fragments of the neurofibromatosis type 1 protein neurofibromin. Protein Expr. Purif. 65:30–37.
45. Angelini A, Tosi T, Mas P, Acajjaoui S, Zanotti G, Terradot L, Hart DJ (2009) Expression of Helicobacter pylori CagA domains by library-based construct screening. FEBS J. 276:816–824.
46. Tarendeau F, Crepin T, Guilligay D, Ruigrok RW, Cusack S, Hart DJ (2008) Host determinant residue lysine 627 lies on the surface of a discrete, folded domain of influenza virus polymerase PB2 subunit. PLoS Pathog 4:e1000136.
47. Guilligay D, Tarendeau F, Resa-Infante P, Coloma R, Crepin T, Sehr P, Lewis J, Ruigrok RW, Ortin J, Hart DJ, Cusack S (2008) The structural basis for cap binding by influenza virus polymerase subunit PB2. Nat Struct Mol Biol 15:500–6.
48. Chesneau A, Yumerefendi H, Hart DJ (2008) The impact of protein expression methodologies on structural proteomics ed Silman I (World Scientific Publishing Company, Singapore).
49. Tarendeau F, Boudet J, Guilligay D, Mas PJ, Bougault CM, Boulo S, Baudin F, Ruigrok RWH, Daigle N, Ellenberg J, Cusack S, Simorre J-P, Hart DJ (2007) Structure and nuclear import function of the C-terminal domain of influenza virus polymerase PB2 subunit. Nat. Struct. Mol. Biol. 14:229–233.
50. Hart DJ, Tarendeau F (2006) Combinatorial library approaches for improving soluble protein expression in Escherichia coli. Acta Crystallogr. D Biol. Crystallogr. 62:19–26.
51. Alzari PM, Berglund H, Berrow NS, Blagova E, Busso D, Cambillau C, Campanacci V, Christodoulou E, Eiler S, Fogg MJ, Folkers G, Geerlof A, Hart D, Haouz A, Herman MD, Macieira S, Nordlund P, Perrakis A, Quevillon-Cheruel S, Tarandeau F, van Tilbeurgh H, Unger T, Luna-Vargas MPA, Velarde M, Willmanns M, Owens RJ (2006) Implementation of semi-automated cloning and prokaryotic expression screening : the impact of SPINE. Acta Crystallogr. D Biol. Crystallogr. 62:1103–1113.
52. Yoshida H, Hensgens CMH, van der Laan JM, Sutherland JD, Hart DJ, Dijkstra BW (2005) An approach to prevent aggregation during the purification and crystallization of wild type acyl coenzyme A : isopenicillin N acyltransferase from Penicillium chrysogenum. Protein Expr. Purif. 41:61–67.
53. Blackburn JM, Hart DJ (2005) Fabrication of protein function microarrays for systems-oriented proteomic analysis. Methods Mol. Biol. Clifton NJ 310:197–216.
54. de Lumley M, Hart DJ, Cooper MA, Symeonides S, Blackburn JM (2004) A biophysical characterisation of factors controlling dimerisation and selectivity in the NF-kappaB and NFAT families. J. Mol. Biol. 339:1059–1075.
55. Boutell JM, Hart DJ, Godber BLJ, Kozlowski RZ, Blackburn JM (2004) Functional protein microarrays for parallel characterisation of p53 mutants. Proteomics 4:1950–1958.
56. Speight RE, Hart DJ, Blackburn JM (2002) Distamycin A affects the stability of NF-kappaB p50-DNA complexes in a sequence-dependent manner. J. Mol. Recognit. JMR 15:19–26.
57. Speight RE, Hart DJ, Sutherland JD, Blackburn JM (2001) A new plasmid display technology for the in vitro selection of functional phenotype-genotype linked proteins. Chem. Biol. 8:951–965.
58. Hart DJ, Speight RE, Sutherland JD, Blackburn JM (2001) Analysis of the NF-kappaB p50 dimer interface by diversity screening. J. Mol. Biol. 310:563–575.
59. Hart DJ, Speight RE, Cooper MA, Sutherland JD, Blackburn JM (1999) The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity. Nucleic Acids Res. 27:1063–1069.