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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. I also
conducted research at the interface between mathematics and musical
composition.
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 Å) 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 Å 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. Plückthun, 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.
Logic of the musical language
The aim of my musical research is to free harmony of the unnecessary
layers of complexity that have accumulated over the centuries and
propose a new logic of the musical language. The results that I
demonstrated, in particular the Theorem of Parsimony, are valid in
diverse contexts, musical or not. They bring (i) new propositions in
the theory of sets, their sequences and combinatorics; and (ii) a
simple and practical approach to create new harmonies and chord
progressions not only in the twelve-tone equal-temperament but also in
unequal temperaments and microtonal systems (66, 67).
References
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
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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
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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
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H. (2000) Biochemistry 39, 1739-1747
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H., and Delepierre, M. (1999) FEBS Lett 446, 81-85
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Biochemistry 40, 7192-7199
33. Bedouelle, H. (2005) Tyrosyl-tRNA synthetases. in
The aminoacyl-tRNA synthetases (Ibba, M., Francklyn, C., and Cusack, S.
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Mol Biol 286, 563-577
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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
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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
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