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Hugues Bedouelle - Research

The research of my laboratory was focused on the relations between the three-dimensional structure of proteins, their conformational stability and their mechanisms of action. We used the multidisciplinary approach of protein engineering and in-vitro molecular evolution.

Main fields of research (from past to present)
Control of genetic expression;
Protein export;
Hybrid proteins;
Recognition between protein and RNA;
Principles for measuring and interpreting protein stability;
Stability and folding of selected proteins;
Recognition between antibody and antigen;
Recognitions between a protective antibody and the four serotypes of dengue virus;
Development of diagnostic tests and vaccines;
Membrane receptors;
Structure of the nucleocapsid of Respiratory Syncytial Virus (RSV);
Reagentless fluorescent biosensors.

Control of genetic expression
The system for the transport of maltose is encoded in Escherichia coli by two divergent operons, under control of the malEp and malKp promoters, and of the transcriptional activators MalT and CRP. I have determined the sequence and functional structure of the region of control that is located between these two operons. In particular, I have determined the DNA sequence of this region (1) and the start-sites of transcription for both operons by sequencing the 5'-triphosphate ends of their messenger RNAs (mRNA), identified the start-sites of translation for the malE and malK genes, and proposed recognition sequences on DNA for MalT and CRP (2). The selection and characterization of mutations enabled me to show that part of the control region is common to both operons whose expressions are therefore coordinated (3). This work was performed while I was a PhD student in M. Hofnung's laboratory.

Threonyl-tRNA synthetase from E. coli represses the translation of its own gene thrS. On the basis of the available data, I proposed that two stem-and-loop structures in the leader mRNA of thrS mimick the anticodon stem-and-loop of tRNA-Thr and bind the two symmetrical binding sites of the latter in the threonyl-tRNA synthetase homodimer (4). This proposal was subsequently shown to be correct.

Protein export
The maltose binding protein (MBP or MalE) is the product of the malE gene in E. coli. It is exported through the cytoplasmic membrane into the periplasmic space by the intermediate of an N-terminal signal peptide. This short peptide (26 residues for MalE) contains a stretch of hydrophobic amino acid residues that drives its insertion into the cytoplasmic membrane and thus initiates export. The signal peptide is subsequently cleaved to release the exported protein into the periplasm. We have determined the DNA sequence of the malE gene and a nearly complete amino acid sequences of the mature form of MalE (in coll. with I. Zabin, USA) (5). Using MalE as a model protein, I performed the first characterization of mutations in a signal peptide that prevent protein export (in coll. with J. Beckwith, USA). I found that the change of a single hydrophobic amino acid to a charged amino acid within the signal peptide is sufficient to block export. One of the changes introduced a proline within the signal peptide and thus constrained its conformation (6). Using this set of well characterized mutations, I attempted an analysis of the relations between conformation and function of signal peptides (7). This work was performed while I was a PhD student in M. Hofnung's laboratory.

I constructed a hybrid protein between MalE and the Klenow polymerase from E. coli and showed that it is exported into the periplasmic space in significant quantities. Thus, a cytoplasmic protein can be exported through a membrane (8).

Hybrid proteins
We performed random insertions of a short DNA linker, encoding a restriction site, into the malE gene of E. coli and thus a first analysis of the relations between structure and function of the maltose binding protein MalE. Some insertions affected the MalE function while others were permissive (9, 10). These permissive sites in MalE were subsequently used by others to insert antigenic peptides and raise immune responses. I used the 3'-terminal insertions to develop the use of MalE as an affinity handle for the purification of foreign proteins and peptides, in particular functional antibody fragments (8, 11-14). Such hybrids allowed us to show that the fusion of an exported protein with a Leucine Zipper induces its dimerization in vivo (15); and that one partner of a hybrid protein can destabilize the other partner (16).

Recognition between protein and RNA
The advent of oligonucleotide site-directed mutagenesis around year 1980, opened new perspectives for the study of proteins and resulted in a new field of research, protein engineering. I contributed to the development of an improved method of oligonucleotide site-directed mutagenesis (17) and then applied it to a problem of recognition between proteins and RNAs. The aminoacyl-tRNA synthetases (aaRS) are the enzymes that translate the genetic code in vivo by attaching each of the 20 amino acids to the cognate transfer RNAs (tRNAs), i.e. those that carry the corresponding anticodon. Thus, tyrosyl-tRNA synthetase (TyrRS) attaches tyrosine (Tyr) to the cognate tRNA-Tyr. The aaRSs have been divided into two classes of 10 elements each.

I studied TyrRS from Bacillus stearothermophilus more particularly. TyrRS is an homodimer. We constructed heterodimeric TyrRSs that carried different mutations in the two subunits, and analyzed their activities. These construction showed that the binding site of one tRNA-Tyr molecule straddles both subunits of TyrRS (18). I then mapped the binding site of tRNA-Tyr on the surface of TyrRS from B. stearothermophilus, whose crystal structure was known, and constructed a structural model of their complex by site-directed mutagenesis and molecular modeling (in coll. with G. Winter, UK) (19, 20). This model showed that TyrRS recognizes its cognate tRNAs in an exceptional way among the 10 aminoacyl-tRNA synthetases of Class I (21, 22) and it was experimentally confirmed by others. Some of the mutations that I constructed, were useful to reconstruct the transition state for the activation of tyrosine by tyrosyl-tRNA synthetase (in coll. with A.R. Fersht) (23).

I discovered that the overproduction of TyrRS is toxic to the host cell. This toxicity likely results from the mischarging of non-cognate tRNAs by TyrRS and thus from the incorporation of tyrosine in place of other amino acids into the cellular proteins (24). We also discovered that some mutations of TyrRS, in the binding site of tRNA-Tyr, render TyrRS toxic to the host cell (24). I used this property to prove the existence of a new mechanism of recognition between macromolecules, by electrostatic repulsion, that participates in the rejection of non-cognate tRNAs by TyrRS (25, 26).

The C-terminal domain (residues 320-419) of TyrRS is disordered in the crystal structure of the free enzyme. We have shown that this C-terminal domain is folded in solution (27-29); determined its three-dimensional structure by Nuclear Magnetic Resonance (in coll. with M. Delepierre) (30, 31); and shown the functional role of the flexible peptide that links the N- and C-terminal domains (32).

I wrote three reviews on tyrosyl-tRNA synthetases, their recognition of the cognate tRNA-Tyr and their discrimination against the non-cognate tRNAs (21, 22, 33).

Principles for measuring and interpreting protein stability
The ability to measure the thermodynamic stability of proteins with precision is important for both academic and applied research. Such measurements rely on mathematical models of the protein denaturation profile, i.e. the relation between a global protein signal, corresponding to the folding states in equilibrium, and the variable value of a denaturing agent, either heat or a chemical molecule, e.g. urea or guanidinium hydrochloride. I reviewed the underlying basic physical laws, and showed in detail how to derive model equations for the unfolding equilibria of homomeric or heteromeric proteins up to trimers and potentially tetramers, with or without folding intermediates. Such equations cannot be derived for pentamers or higher oligomers (34). We expanded the method to signals that do not correspond to extensive protein properties (34-36). I reviewed and expanded methods: (i) for uncovering hidden intermediates of unfolding; (ii) for comparing and interpreting the thermodynamic parameters that derive from stability measurements for cognate wild-type and mutant proteins (34). This work provided a robust theoretical basis for measuring the stability of complex proteins.

Stability and folding of selected proteins
TyrRS from B. stearothermophilus is a dimeric protein. We have shown that it unfolds through a monomeric intermediate and quantified its thermodynamic stability. This stability is very high and has three additive components: the stability of the association between the two subunits, and the stabilities of the two identical subunits (37). Thus, dimeric proteins may have an advantage over monomeric proteins in terms of stability.

E. coli is a mesophilic bacterium whereas B. stearothermophilus is a thermophile. The TyrRSs from E. coli and B. stearothermophilus are 56% identical in amino acid sequence. To understand the molecular bases of their difference in stability, we made hybrid proteins between the two TyrRSs by in vivo recombinations between their genes, in a kind of in-vivo DNA shuffling. The hybrid TyrRSs were active, which showed that the sequences and structures of the two parental proteins can replace each other locally and still give an active TyrRS enzyme. Analysis of the stabilities of the hybrids showed that the greater stability of TyrRS from B. stearothermophilus is due to cumulative changes of residues scattered along the sequence (38). We also identified a cluster of interacting residues in the structural core of TyrRS from B. stearothermophilus and changed its residues into the corresponding residues of TyrRS from E. coli, either individually or in groups. Some mutations affected the stability of the association between the subunits at a distance, whereas others affected the stability of the protomer. The effects of the mutations on stability could depend on their pathway of introduction: for example, the effects of mutations 1 and 2 on stability could be neutral when introduced in this order whereas mutation 2 was destabilizing and mutation 1 stabilizing when introduced in this reverse order. Thus, the effect of a mutation on stability may depend on its structural context and evolution may take preferential pathways (35).

We have developed a method of molecular design, based on the consensus of sequence, to strongly improve the stability of a variable fragment scFv of antibody by mutations. We quantified the contributions of resistance to denaturation and cooperativity of unfolding to the variations of stability caused by the mutations, and showed that the variations of cooperativity involved residual structures of the denatured state (39).

The Flavivirus genus includes severe human pathogens, e.g. the four serotypes of dengue virus (DENV1 to DENV4), the yellow fever virus, the Japanese encephalitis virus and West Nile virus. Domain III (ED3) of the viral envelope (E) protein interacts with cellular receptors and includes epitopes that are recognized by neutralizing antibodies. We have measured the thermodynamic stabilities of recombinant ED3 domains from the above viruses. An ED3 domain, rationally designed and possessing the consensus sequence of numerous DENV strains coming from the four serotypes, was highly stable. The stability of an ED3 domain was increased by design without affecting its reactivity towards human serums that were infected by the corresponding virus. The Tm (temperature of half-unfolding) of ED3 was higher than 69 C for all the tested viruses and reached 86 C for the consensus ED3 domain.  These results may allow one to better understand and manipulate the stability properties of ED3 for its use as diagnostic, vaccine or therapeutic tools (40).

Recognition between antibody and antigen
The crystal structure of the complex between monoclonal antibody mAbD1.3 and its antigen, hen egg-white lysozyme, is available. We have shown by mutagenesis that a limited subset of the physical contacts between mAbD1.3 and lysozyme contributes to the kinetics and energy of their interaction, and it involves both hydrophobic and charged residues (41). Moreover, we have shown that the improvement of affinity (60-fold) of mAbD1.3 during the maturation of the immune response is due to only one among the five somatic hypermutations that are present (42). Using mAbD1.3 as a model, we have shown that the rate of dissociation between an antibody and a protein antigen determines the efficacy with which the antibody serves as an intermediate for the presentation of its antigen to T-cells (in coll. with C. Leclerc) (43).

We have analyzed the cross-reactivities of a monoclonal antibody, mAb164, towards a protein, TrpB2, and a mimetic undecapeptide derivative, P11, by mutagenesis of the two forms of the antigen. We have thus shown that the isolated and integrated forms of the epitope are recognized by the antibody in the same loop conformation and by the same structural mechanism (13, 44).

We have improved the affinity and protective power of a non-human primate antibody that is directed against the anthrax lethal toxin by a new method of in-vitro directed evolution. This method targets the hypervariable loops (CDR, complementarity determining region) of the antibody simultaneously and exclusively, and therefore does not modify its immunogenicity (45). Moreover, we have modified the scaffold of this antibody to make its sequence closer to those of human germinal gene segments, by in-silico and in-vitro engineering. This germinal humanization does not modify the affinity of an antibody for its antigen and brings its immunogenicity below those of fully human antibodies for clinical applications (in coll. with P. Thullier) (46).

Recognitions between a protective antibody and the four serotypes of dengue virus
Dengue is a viral disease that affects 100 millions of persons per year and can be fatal. The envelope (E) protein of the virus comprises three domains ED1 to ED3, and a transmembrane segment (see above). Monoclonal antibody mAb4E11 neutralizes the four serotypes of dengue virus. We have mapped the energetic paratope (binding site of the antigen) of mAb4E11 by mutagenesis, shown that its diversity and junction residues constitute hot-spots of binding energy for the interaction with the antigen, and thus that it exists a direct link between the generations of diversity and affinity in antibodies (47). We have mapped the energetic epitope of mAb4E11 in the ED3 domain by mutagenesis. We have shown that it is discontinuous, constitutes an antigenic signature for the group of the dengue viruses, and can be transplanted in the E protein of non-cognate Flaviviruses (48, 49).

We have determined the crystal structures at high resolution (between 1.6 and 2.1 ) of the complexes between the scFv variable fragment of mAb4E11 and each of the four serotypes of the ED3 domain. These structures have revealed the essential determinants of the cross-reactivities towards the four serotypes. They have explained the wide neutralization spectrum of mAb4E11 despite important differences in affinities between its Fab fragment and the four ED3 serotypes (in coll. with F.A. Rey) (50). Through a thorough analysis of the structures, we have shown the existence of indirect contacts between mAb4E11 and ED3, mediated by water molecules and conserved between serotypes. The third complementarity determining region of the light chain (L-CDR3) of mAb4E11 does not contact ED3. Through the construction of additional mutations in both ED3 and the Fab fragment of mAb4E11, we have shown that the effects of (hyper)-mutations in L-CDR3 on affinity are caused by conformational changes and indirect interactions with ED3 through other CDRs. We have exchanged residues between ED3 serotypes and shown that their effects on affinity are context dependent. This research has shown that conformational changes, structural context, and indirect interactions should be considered when studying the cross-reactivities between antibodies and different serotypes of viral antigens, for a better design of diagnostics, vaccine, and therapeutic tools (51).

Development of diagnostic tests and vaccines
During an internship in M. Hofnung's laboratory in 1975-76, I contributed to the adaptation of a genotoxicity test (the Ames test) to the mineral oils (52, 53).

The IgMs are the first immunoglobulins that appear during a viral infection, they are pentameric and have low affinities. We have developed families of recombinant antigens to simplify the early serological tests of infections by Flaviviruses. These reagents are based on artificial homomultimers of domain ED3 from protein E, and use the phenomenon of avidity to increase their energy of interaction with IgMs. One of these families is based on the construction of hybrids, at the genetic level, between ED3 and an improved alkaline phosphatase from E. coli, which is a periplasmic dimeric protein. Such reagents can be constructed and produced rapidly in E. coli for any emerging Flavivirus. They make MAC-ELISA assays (MAC, IgM Antibody Capture; ELISA, Enzyme Linked ImmunoSorbent Assay) more sensitive and simpler to perform.

Infection by one of the four serotypes DENV1 to DENV4 of dengue virus induces a long lasting protection against this serotype but not against the three other serotypes. A subsequent infection by a different serotype is a risk factor for a severe dengue. We have determined the serotype specificities and cross-reactivities of human IgMs, directed against ED3, by using a collection of well-characterized human serums that were infected or non-infected by DENV. The recognitions of the four serotypes of ED3 by the serums were tested with the above MAC-ELISA assays and statistically analyzed with ROC (Receiving Operator Characteristic) curves. The DENV infected serums contained IgMs that reacted with one or several serotypes of ED3. The discrimination by ED3 between serums infected by the homotypic DENV and non-infected serums varied with the serotype in the decreasing order DENV1 >DENV2 >DENV3 >DENV4. The ED3 domain of DENV1 behaved as a universal antigen for the detection of IgMs against the four serotypes of DENV. Some serotypes of ED3 discriminated between IgMs directed against the homotypic DENV and heterotypic DENVs. These data may allow one to better understand the IgM response and protection against DENV since ED3 is used as an antigen in diagnostic assays and as an immunogen in vaccine candidates (in coll. with Ph. Dussart) (54).

I have contributed to the successfull evaluation of a vaccine against dengue, based on a hybrid virus between the live attenuated vaccine against measles and minimal domains of the prM and E proteins from the four serotypes of dengue virus (in coll. with F. Tangy) (55, 56).

Membrane receptors
Ribosomal Protein SA (RPSA or LamR1) belongs to the ribosome but is also a membrane receptor for laminin, growth factors, prion, pathogenic agents and the anticarcinogen epigallocatechine-gallate (EGCG, a constituent of green tea). RPSA contributes to the crossing of the hemato-encephalitic barrier by neurotropic viruses and bacteria, and is a marker of metastasis. The human RPSA includes an N-terminal domain, which is homologous to the prokaryotic ribosomal protein S2, and a C-terminal extension, which is conserved in vertebrates. We have shown that the N-terminal domain unfolds according to an equilibrium between three monomeric states, the intermediate of unfolding is predominant at the body temperature of 37 C, and the C-terminal domain is intrinsically disordered (57).

We have constructed recombinant derivatives of RPSA and quantified their interactions with ligands by immunochemical and spectrofluorometric methods in vitro. These studies have shown that both N- and C-terminal domains of RPSA bind laminin with dissociation constants (Kd) of 300 nM. Heparin binds only to the N-terminal domain and competes with the negatively charged C-terminal domain for binding to laminin. Thus, the C-terminal domain mimicks heparin. EGCG binds only to the N-terminal domain with a Kd of 100 nM. The ED3 domains from the envelope proteins of the yellow fever virus and serotypes 1 and 2 of dengue virus preferentially bind to the C-terminal domain whereas that from the West-Nile virus binds only to the N-terminal domain. This in-vitro quantitative approach may help identify the mechanisms of RPSA action and thus fight against cancer and some infectious agents (58). Our work on RPSA/LamR1 solved some paradox in the literature on this protein, introduced quantitative methods in its study and clarified the latter.

The natural killer (NK) cells contribute to the innate immune response. The NKp44 receptor is present at the surface of the NK cells and is one of the activators of these cells. We have studied the interaction between the NK cells and two Flaviviruses, the dengue virus (DENV) and the West-Nile virus (WNV). We have shown that the NKp44 receptor participates in the activation of NK cells by these two viruses through a direct interaction with protein E, and more specifically with the ED3 domain for WNV. The activation of NK cells requires only their exposure to the virus and not their infection (in coll. with A. Porgador, Israel) (59).

Structure of the nucleocapsid of Respiratory Syncytial Virus (RSV)
I have contributed to the resolution of the structure for the helical nucleocapsid of RSV, the most important pathogen of the lower respiratory tract in children. The crystal structure at 3 resolution consists of a disk that includes a random RNA and 10 protomers of the nucleoprotein (in coll. with F. Rey) (60).

Reagentless fluorescent biosensors
I have developed original approaches to transform any element of a natural or artificial family of antigen binding proteins (AgBP) into a reagentless fluorescent (RF) biosensor. They consist in introducing a unique residue of cysteine in the neighbourhood of the AgBP paratope and then in coupling a solvatochromic fluorophore to this cysteine. The binding of the antigen protects the fluorescent group against an extinction by the solvent and results in a variation of fluorescence. We have described and validated rules of design to choose the coupling site, based either on the three-dimensional structure of the complex between AgBP and antigen when known (61, 62), or on mutagenesis data when unknown (63, 64), with a high success rate. I have theoretically defined the sensitivity of the RF biosensors and we have shown that their sensitivity and dynamic interval can be improved by molecular design (65). We have validated these approaches with antibodies and artificial proteins, either Designed Ankyrin Repeat Proteins (DARPins; in coll. with A. Plckthun, Switzerland) or OB-fold proteins (Affitin or Nanofitin, in coll. with F. Pecorari). These artificial proteins have excellent properties of recombinant production and stability. The RF biosensors have potential applications in research, health, environment, defense and industry.

1.    Bedouelle, H., and Hofnung, M. (1982) Mol Gen Genet 185, 82-87
2.    Bedouelle, H., Schmeissner, U., Hofnung, M., and Rosenberg, M. (1982) J Mol Biol 161, 519-531
3.    Bedouelle, H. (1983) J Mol Biol 170, 861-882
4.    Bedouelle, H. (1993) J Mol Biol 230, 704-708
5.    Duplay, P., Bedouelle, H., Fowler, A., Zabin, I., Saurin, W., et al. (1984) J Biol Chem 259, 10606-10613
6.    Bedouelle, H., Bassford, P. J., Jr., Fowler, A. V., Zabin, I., Beckwith, J., et al. (1980) Nature 285, 78-81
7.    Bedouelle, H., and Hofnung, M. (1981) Prog Clin Biol Res 63, 399-403
8.    Bedouelle, H., and Duplay, P. (1988) Eur J Biochem 171, 541-549
9.    Duplay, P., Bedouelle, H., Szmelcman, S., and Hofnung, M. (1985) Biochimie 67, 849-851
10.    Duplay, P., Szmelcman, S., Bedouelle, H., and Hofnung, M. (1987) J Mol Biol 194, 663-673
11.    Blondel, A., and Bedouelle, H. (1990) Eur J Biochem 193, 325-330
12.    Bregegere, F., Schwartz, J., and Bedouelle, H. (1994) Protein Eng 7, 271-280
13.    Rondard, P., Bregegere, F., Lecroisey, A., Delepierre, M., and Bedouelle, H. (1997) Biochemistry 36, 8954-8961
14.    Bedouelle, H., Renard, M., Belkadi, L., and England, P. (2002) Res Microbiol 153, 395-398
15.    Blondel, A., and Bedouelle, H. (1991) Protein Eng 4, 457-461
16.    Blondel, A., Nageotte, R., and Bedouelle, H. (1996) Protein Eng 9, 231-238
17.    Carter, P., Bedouelle, H., and Winter, G. (1985) Nucleic Acids Res 13, 4431-4443
18.    Carter, P., Bedouelle, H., and Winter, G. (1986) Proc Natl Acad Sci U S A 83, 1189-1192
19.    Bedouelle, H., and Winter, G. (1986) Nature 320, 371-373
20.    Labouze, E., and Bedouelle, H. (1989) J Mol Biol 205, 729-735
21.    Bedouelle, H. (1990) Biochimie 72, 589-598
22.    Bedouelle, H., Guez-Ivanier, V., and Nageotte, R. (1993) Biochimie 75, 1099-1108
23.    Fersht, A. R., Knill-Jones, J. W., Bedouelle, H., and Winter, G. (1988) Biochemistry 27, 1581-1587
24.    Bedouelle, H., Guez, V., Vidal-Cros, A., and Hermann, M. (1990) J Bacteriol 172, 3940-3945
25.    Vidal-Cros, A., and Bedouelle, H. (1992) J Mol Biol 223, 801-810
26.    Bedouelle, H., and Nageotte, R. (1995) Embo J 14, 2945-2950
27.    Guez-Ivanier, V., and Bedouelle, H. (1996) J Mol Biol 255, 110-120
28.    Jermutus, L., Guez, V., and Bedouelle, H. (1999) Biochimie 81, 235-244
29.    Guez, V., Nair, S., Chaffotte, A., and Bedouelle, H. (2000) Biochemistry 39, 1739-1747
30.    Pintar, A., Guez, V., Castagne, C., Bedouelle, H., and Delepierre, M. (1999) FEBS Lett 446, 81-85
31.    Guijarro, J. I., Pintar, A., Prochnicka-Chalufour, A., Guez, V., Gilquin, B., et al. (2002) Structure 10, 311-317
32.    Gaillard, C., and Bedouelle, H. (2001) Biochemistry 40, 7192-7199
33.    Bedouelle, H. (2005) Tyrosyl-tRNA synthetases. in The aminoacyl-tRNA synthetases (Ibba, M., Francklyn, C., and Cusack, S. eds.), Landes Bioscience. pp 111-124
34.    Bedouelle, H. (2016) Biochimie 121, 29-37
35.    Park, Y. C., Guez, V., and Bedouelle, H. (1999) J Mol Biol 286, 563-577
36.    Monsellier, E., and Bedouelle, H. (2005) Protein Eng Des Sel 18, 445-456
37.    Park, Y. C., and Bedouelle, H. (1998) J Biol Chem 273, 18052-18059
38.    Guez-Ivanier, V., Hermann, M., Baldwin, D., and Bedouelle, H. (1993) J Mol Biol 234, 209-221
39.    Monsellier, E., and Bedouelle, H. (2006) J Mol Biol 362, 580-593
40.    Zidane, N., Dussart, P., Bremand, L., Villani, M. E., and Bedouelle, H. (2013) Protein Eng Des Sel 26, 389-399
41.    England, P., Bregegere, F., and Bedouelle, H. (1997) Biochemistry 36, 164-172
42.    England, P., Nageotte, R., Renard, M., Page, A. L., and Bedouelle, H. (1999) J Immunol 162, 2129-2136
43.    Guermonprez, P., England, P., Bedouelle, H., and Leclerc, C. (1998) J Immunol 161, 4542-4548
44.    Rondard, P., Goldberg, M. E., and Bedouelle, H. (1997) Biochemistry 36, 8962-8968
45.    Laffly, E., Pelat, T., Cedrone, F., Blesa, S., Bedouelle, H., et al. (2008) J Mol Biol 378, 1094-1103
46.    Pelat, T., Bedouelle, H., Rees, A. R., Crennell, S. J., Lefranc, M. P., et al. (2008) J Mol Biol 384, 1400-1407
47.    Bedouelle, H., Belkadi, L., England, P., Guijarro, J. I., Lisova, O., et al. (2006) Febs J 273, 34-46
48.    Thullier, P., Demangel, C., Bedouelle, H., Megret, F., Jouan, A., et al. (2001) J Gen Virol 82, 1885-1892
49.    Lisova, O., Hardy, F., Petit, V., and Bedouelle, H. (2007) J Gen Virol 88, 2387-2397
50.    Cockburn, J. J., Navarro Sanchez, M. E., Fretes, N., Urvoas, A., Staropoli, I., et al. (2012) Structure 20, 303-314
51.    Lisova, O., Belkadi, L., and Bedouelle, H. (2014) J Mol Recognit 27, 205-214
52.    Bedouelle, H., and Hofnung, M. (1978) C R Acad Sci Hebd Seances Acad Sci D 287, 891-894
53.    Hermann, M., Chaude, O., Weill, N., Bedouelle, H., and Hofnung, M. (1980) Mutat Res 77, 327-339
54.    Zidane, N., Dussart, P., Bremand, L., and Bedouelle, H. (2013) BMC Infect Dis 13, 302
55.    Brandler, S., Lucas-Hourani, M., Moris, A., Frenkiel, M. P., Combredet, C., et al. (2007) PLoS Negl Trop Dis 1, e96
56.    Brandler, S., Ruffie, C., Najburg, V., Frenkiel, M. P., Bedouelle, H., et al. (2010) Vaccine 28, 6730-6739
57.    Ould-Abeih, M. B., Petit-Topin, I., Zidane, N., Baron, B., and Bedouelle, H. (2012) Biochemistry 51, 4807-4821
58.    Zidane, N., Ould-Abeih, M. B., Petit-Topin, I., and Bedouelle, H. (2013) Biosci Rep 33, 113-124
59.    Hershkovitz, O., Rosental, B., Rosenberg, L. A., Navarro-Sanchez, M. E., Jivov, S., et al. (2009) J Immunol 183, 2610-2621
60.    Tawar, R. G., Duquerroy, S., Vonrhein, C., Varela, P. F., Damier-Piolle, L., et al. (2009) Science 326, 1279-1283
61.    Renard, M., Belkadi, L., Hugo, N., England, P., Altschuh, D., et al. (2002) J Mol Biol 318, 429-442
62.    Brient-Litzler, E., Pluckthun, A., and Bedouelle, H. (2010) Protein Eng Des Sel 23, 229-241
63.    Renard, M., Belkadi, L., and Bedouelle, H. (2003) J Mol Biol 326, 167-175
64.    Miranda, F. F., Brient-Litzler, E., Zidane, N., Pecorari, F., and Bedouelle, H. (2011) Biosens Bioelectron 26, 4184-4190
65.    Renard, M., and Bedouelle, H. (2004) Biochemistry 43, 15453-15462