Download Surface plasmon resonance as a tool in the

Ph. D. Thesis
Ph. D. Program: Fundamental Chemistry,
Organic Chemistry, 1997-1999.
Surface plasmon resonance as a tool in the
functional analysis of an immunodominant site
in foot-and-mouth disease virus
Paula Alexandra de Carvalho Gomes
Department of Organic Chemistry, Faculty of Chemistry.
Division of Experimental and Mathematical Sciences.
University of Barcelona, 2000
Department of Organic Chemistry, Faculty of Chemistry.
Division of Experimental and Mathematical Sciences.
University of Barcelona, 2000
Dissertation presented by
Paula Alexandra de Carvalho Gomes
to apply for the degree of Doctor in Chemical Sciences
at the University of Barcelona
and revised by
Dr. David Andreu
En primer lugar, me gustaría agradecer al Dr. David Andreu, supervisor de la presente tesis, y al Dr.
Ernest Giralt por la calurosa acogida en su laboratorio, dándome la gran oportunidad de formar parte
de su grupo de investigación. También quisiera agradecerles las fructuosas discusiones y el constante
apoyo siempre que me sentía perdida entre “biacores” y péptidos...
A Jaume y Chary, de los SCT, porque siempre han traído un rayo de Sol a mis días de autismo cuando
tocaba hacer biacore (al final, la “unidad” paula se puede desacoplar del BIACORE 1000!)
Estoy igualmente en deuda con Wendy Fernández, Dr. Núria Verdaguer y Dr. Ignacio Fita por
presentarme el guapísimo mundo de la cristalografía de proteínas y por toda la paciencia que tuvieron
guiando mis pasitos infantiles en este campo de investigación.
A todo el maravilloso “grup 10”, mi casa y mi familia en estos cuatro años en que Barcelona ha sido mi
ciudad adoptiva...quisiera agradecer muy especialmente a los compañeros y compañeras que han
conquistado un rincón especial en mi corazón: Judit(h) “Peter Pan”, Lorena (la dulce), Meritxell (alias,
Cielitos), Albert(it)o Adeva, Melenita, Marta Molina, Mac… y jamás olvidaré a gente como Oscar
Millet, genio por profesión… y ornitólogo por devoción… y a Paul, por su formidable personalidad.
Antes de mais devo agradecer à Prof. Doutora Maria Joaquina Amaral Trigo, não só por me apoiar e
acreditar em mim profissionalmente, mas também pela sua valiosa amizade.
Agradeço à Fundação Calouste Gulbenkian (Lisboa, Portugal) a bolsa de doutoramento que me foi
concedida, o apoio financeiro para assistir a congressos internacionais e a gentileza com que sempre
fui tratada.
À Lia, catalã por motivos geográfico-sentimentais, portuguesa pelo idioma (si beim qui teim um cerrto
sôtaqui dá cidádji dji São Paulo, né?) e com a bondade e a doçura de brasileira que é. És daquelas
jóias raras com quem se pode sempre contar.
Aos meus amigos de sempre, Helena, Carla e Alfredo, por continuarmos a ser uma espécie de
D’Artagnan e os, ou melhor, as Três Mosqueteiras...”voltar” não seria o mesmo sem vocês e nunca me
cansarei de agradecer a vossa amizade!
Aos meus pais, Tina e Quim, e ao Richard, por
tudo o que são para mim, dedico a presente Tese.
General index
Abstract
Resumen
Abbreviations
Amino acids
Amino acid protecting groups
Resins, handles and coupling reagents
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0. Introduction
Surface plasmon resonance biosensors
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0.1 Surface plasmon resonance
0.2 Real-time biospecific interaction analysis
0.3 Measuring kinetics of biospecific interactions
References
Foot-and-mouth disease virus
0.4 Foot-and-mouth disease
0.5 Foot-and-mouth disease virus
0.6 The development of anti-FMDV vaccines
References
Objectives
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1. SPR screening of synthetic peptides from the GH loop of FMDV
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2. Antigenic determinants in the GH loop of FMDV C1-Barcelona (or C-S30)
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1.0 Introduction
1.1 Optimisation of the experimental set-up
1.2 Application to the systematic screening of FMDV peptides
1.3 Use of other site A – directed monoclonal antibodies
1.4 Probing subtle differences in peptide and mAb behaviour by SPR
1.5 Validity of the experimental kinetic constants
1.6 Relevance of the SPR data for FMDV studies
References
2.0 Introduction
2.1 Peptides mimicking the GH loop of FMDV C1-Barcelona and the
corresponding partial mutants
2.2 SPR study of the C-S30 peptides
2.3 Competition ELISA analysis of the C-S30 pentadecapeptides
2.4 Size effects in the antigenicity of C-S30 peptides
2.5 Input from parallel X-ray diffraction studies
2.6 Effect of conformation in the antigenicity of C-S30 peptides
2.7 Antigenic evaluation of C-S30 peptides through solution affinity SPR analysis
2.8 Two-dimensional proton nuclear magnetic resonance studies of C-S30 peptides
2.9 Recapitulation
References
3. Antigenic peptides with non-natural replacements within the GH loop of FMDV
3.0 Introduction
3.1 Peptides that combine antigenicity-enhancing replacements in the GH loop
3.2 Direct kinetic SPR analysis
3.3 Indirect SPR kinetic analysis using a high molecular weight competitor antigen
3.4 Solution affinity SPR analysis of the peptide antigens
3.5 Two-dimensional 1H-NMR analysis of peptide A15(FPS)
3.6 X-ray diffraction crystallography analysis of a peptide-antibody complex
3.7 Recapitulation
References
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144
145
i
Conclusions
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4. Materials & Methods
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4.1 General procedures
4.1.1 Solvents and chemicals
4.1.2 Instrumentation
4.1.3 Analytical methods
4.1.4 Chromatographic methods
References
4.2 Solid-phase peptide synthesis
4.2.1 Solid-phase peptide synthesis protocols
4.2.2 Synthesis of peptides from the GH loop of FMDV
References
4.3 Antigenic evaluation of the FMDV peptides
4.3.1 SPR analysis of peptide-antibody interactions
4.3.2 Enzyme-linked immunosorbent assays
References
4.4 Structural studies of the FMDV peptides
4.4.1 Two-dimensional proton nuclear magnetic resonance
4.4.2 Protein X-ray diffraction crystallography
References
ii
Abstract
A fast and direct surface plasmon resonance (SPR) method for the kinetic analysis of the interactions between
peptide antigens and immobilised monoclonal antibodies (mAb) has been established. Protocols have been
developed to overcome the problems posed by the small size of the analytes (< 1600 Da). The interactions
were well described by a simple 1:1 bimolecular interaction and the rate constants were self-consistent and
reproducible. The key features for the accuracy of the kinetic constants measured were high buffer flow rates,
medium antibody surface densities and high peptide concentrations. The method was applied to an extensive
analysis of over 40 peptide analogues towards two distinct anti-FMDV antibodies, providing data in total
agreement with previous competition ELISA experiments.
Eleven linear 15-residue synthetic peptides, reproducing all possible combinations of the four replacements
found in foot-and-mouth disease virus (FMDV) field isolate C-S30, were evaluated. The direct kinetic SPR
analysis of the interactions between these peptides and three anti-site A mAbs suggested additivity in all
combinations of the four relevant mutations, which was confirmed by parallel ELISA analysis. The four-point
mutant peptide (A15S30) reproducing site A from the C-S30 strain was the least antigenic of the set, in
disagreement with previously reported studies with the virus isolate. Increasing peptide size from 15 to 21
residues did not significantly improve antigenicity. Overnight incubation of A15S30 with mAb 4C4 in solution
showed a marked increase in peptide antigenicity not observed for other peptide analogues, suggesting that
conformational rearrangement could lead to a stable peptide-antibody complex. In fact, peptide cyclization
clearly improved antigenicity, confirming an antigenic reversion in a multiply substituted peptide. Solution NMR
studies of both linear and cyclic versions of the antigenic loop of FMDV C-S30 showed that structural features
previously correlated with antigenicity were more pronounced in the cyclic peptide.
Twenty-six synthetic peptides, corresponding to all possible combinations of five single-point antigenicityenhancing replacements in the GH loop of FMDV C-S8c1, were also studied. SPR kinetic screening of these
peptides was not possible due to problems mainly related to the high mAb affinities displayed by these synthetic
antigens. Solution affinity SPR analysis was employed and affinities displayed were generally comparable to or
even higher than those corresponding to the C-S8c1 reference peptide A15. The NMR characterisation of one
of these multiple mutants in solution showed that it had a conformational behaviour quite similar to that of the
native sequence A15 and the X-ray diffraction crystallographic analysis of the peptide – mAb 4C4 complex
showed paratope – epitope interactions identical to all FMDV peptide – mAb complexes studied so far. Key
residues for these interactions are those directly involved in epitope – paratope contacts (141Arg,
143
Asp,
146
His)
as well as residues able to stabilise a particular peptide global folding. A quasi-cyclic conformation is held up by
a hydrophobic cavity defined by residues 138, 144 and 147 and by other key intrapeptide hydrogen bonds,
delineating an open turn at positions 141, 142 and 143 (corresponding to the Arg-Gly-Asp motif).
iii
Resumen
Se diseñó un método rápido y sencillo para el análisis cinético por resonancia de plasmón superficial (RPS) de
las interacciones entre antígenos peptídicos de bajo peso molecular (< 1600 Da) y anticuerpos monoclonales
(AM) inmovilizados en la superficie de un chip sensor. Dichas interacciones se ajustaron a un modelo de
interacción bimolecular 1:1 y las constantes cinéticas obtenidas resultaron fiables y reproducibles. Los
parámetros clave para la calidad de las constantes cinéticas medidas fueron un flujo de tampón elevado, una
densidad superficial de AM intermedia y una elevada concentración de péptido. El método se extendió a más
de 40 análogos peptídicos frente a dos AM contra el virus de la fiebre aftosa (VFA), obteniéndose total
correlación con datos anteriores de ELISA competitivo.
Se sintetizaron once pentadecapéptidos con todas las combinaciones posibles de las cuatro mutaciones que
caracterizan el bucle GH del aislado C-S30 del VFA respecto a la secuencia de referencia C-S8c1. Los
resultados del análisis cinético directo, por RPS, de la antigenicidad de estos péptidos frente a tres AM
sugirieron que dichas combinaciones eran aditivas, observación que fué confirmada por ELISA competitivo.
Así, el tetramutante (A15S30) que mimetiza el bucle GH de C-S30 resultó ser el peor antígeno de la serie, en
contraste con resultados anteriores con este aislado. Aumentando el tamaño del tetramutante de 15 a 21
aminoácidos no afectó significativamente su antigenicidad. En cambio, una incubación prolongada con el AM
llevó a un aumento de reactividad no observado para otros análogos. Posiblemente una reordenación
conformacional del péptido pudo conllevar a la formación de un complejo estable con el anticuerpo.
Experimentos de RPS con un análogo cíclico del péptido A15S30 confirmaron una reversión en la
antigenicidad del tetramutante inducible a través de restricciones conformacionales. Estudios de ambos
péptidos, lineal y cíclico, por resonancia magnética nuclear (RMN) mostraron que características estructurales
anteriormente correlacionadas con la antigenicidad eran más pronunciadas en el análogo cíclico.
Se prepararon veintiseis péptidos con todas las posibles combinaciones de cinco sustituciones específicas en el
bucle GH del VFA C-S8c1. Dichas sustituciones individuales habían sido objeto de estudios anteriores,
obteniéndose una elevada antigenicidad para los correspondientes péptidos mutantes frente a AM anti-VFA.
No se pudo sistematizar el análisis cinético por RPS de los nuevos mutantes multiples, debido a problemas
tanto en la determinación de las constantes cinéticas de disociación, como en la regeneración de las superficies
de AM. Se utilizó así la RPS para la determinación de la afinidad péptido – AM en solución, obteniéndose
antigenicidades comparables o incluso superiores a las del péptido nativo A15 (VFA C-S8c1). Se estudió uno
de los mutantes multiples (A15FPS) por RMN, observándose una conformación identica a la del péptido
nativo. El estudio del complejo cristalino entre el péptido A15FPS y el AM 4C4 por difracción de RX mostró
que las interacciones parátopo – epítopo eran similares a las observadas con el péptido nativo. Se concluyió
que los residuos clave para el reconocimiento son tanto aquellos involucrados en contactos directos (141Arg,
143
Asp,
146
His) como aquellos que estabilizan el plegamiento adecuado del péptido. Así, una conformación casi
cíclica es soportada por una cavidad hidrofóbica definida por los residuos 138, 144 y 147 y por puentes de
hidrógeno intra-peptídicos clave, diseñándose un bucle abierto centrado en las posiciones 141, 142 and 143
(triplete Arg-Gly-Asp).
iv
Abbreviations
AA
Amino acid
AAA
Amino acid analysis
AcOH
Acetic acid
AM
2-[4-aminomethyl-(2,4-dimethoxyphenyl)phenoxy]acetic acid
APS
Ammonium persulphate
ATR
Attenuated total reflection
BSA
Bovine serum albumin
CDR
Complementarity determining region
Da
Dalton
DCM
Dichloromethane
DIEA
Diisopropylethylamine
DIP
Diisopropylcarbodiimide
DMF
dimethylformamide
EDC
N-ethyl-N’-(dimethylaminopropyl)carbodiimide
EDTA
Ethylenediaminotetraacetic acid
ELISA
Enzyme-linked immunosorbent assay
eq
equivalent
ESI
Electro-spray ionisation
Fab
Fragment, antigen-binding
Fc
Fragment, crystallisable
FMD
Foot-and-mouth disease
FMDV
Foot-and-mouth disease virus
FT-IR
Fourier-transform infrared spectroscopy
HBcAg
Hepatitis B core antigen
HCA
Human carbonic anhydrase
HEPES
4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid
HOBt
1-Hydroxybenzotriazole
HPLC
High performance liquid chromatography
HRV
Human rhino virus
HS
Heparan sulphate
IC50
Antigen concentration giving 50% inhibition
IFC
Integrated fluidic cartridge
Ig
Immunoglobulin
ka
Association rate constant / M-1s-1
KA
Affinity constant (association) / M-1
kd
Dissociation rate constant / s-1
KD
Affinity constant (dissociation) / M
KLH
Keyhole limpet hemocyanin
ks
Apparent/global rate constant / M-1s-1
LED
Light-emitting diode
v
mAb
Monoclonal antibody
MALDI-TOF Matrix-assisted laser desorption ionisation – time-of-flight
MAP
Multiple antigenic peptide
MBHA
p-methylbenzhydrylamine resin
MBS
m-maleimidobenzoyl-N-hydroxysuccinimide
MeCN
acetonitrile
MeOH
methanol
MPLC
Medium-pressure liquid chromatography
MS
Mass spectrometry
MW
Molecular weight
NHS
N-hydroxysuccinimide
NMM
N-methylmorpholine
NMP
N-methylpyrrolidone
NMR
Nuclear magnetic resonance
NOE
Nuclear Overhauser effect
NOESY
Nuclear Overhauser effect spectroscopy
OD
Optical density
PBS
Phosphate buffer saline
PEG
Polyethylene glycol
PS
Polystyrene
PVC
Polyvinyl chloride
R
Response
Req
Response at equilibrium
RI
Refractive index
Rmax
Maximal response
RNA
Ribonucleic acid
Rtot
Total response
RU
Resonance unit
SD
Standard deviation
SDS
Sodium dodecylsulphate
SDS-PAGE
Sodium dodecylsulphate – polyacrylamide gel electrophoresis
SPPS
Solid-phase peptide synthesis
SPR
Surface plasmon resonance
SPW
Surface plasmon wave
VP
Viral protein
TBTU
N-[(1H-benzotriazol-1-yl)dimethylaminomethylene]-N-methylmethaneaminium N-oxide
tetrafluoroborate
TEMED
N,N,N’,N’-tetramethylethylenediamine
TFA
Trifluoroacetic acid
TFE
2,2,2-trifluoroethanol
TIR
Total internal reflection
TOCSY
Total correlation spectroscopy
UV - Vis
Ultraviolet – visible spectroscopy
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Amino acids
Threeletter
One-letter
code
code
Ala
A
Name
Formula
Alanine
CH3
NH CH
Arg
Asn
R
N
Arginine
Asparagine
H2C
(CH2)2
NH CH
CO
CO
NH C NH2
NH
H2C CONH2
NH CH
Asp
D
Aspartic acid
CO
H2C COOH
NH CH
Cys
C
Cysteine
CO
H2C SH
NH CH
Gln
Q
Glutamine
H2C CH2
NH CH
Glu
E
Glutamic acid
G
Glycine
CONH2
CO
H2C CH2
NH CH
Gly
CO
COOH
CO
NH CH2 CO
N
His
H
Histidine
H2C
NH
HN CH CO
Ile
I
Isoleucine
CH(CH3)CH2CH3
NH CH
Leu
L
Leucine
CO
CH2CH(CH3)2
NH CH CO
Lys
K
Lysine
CH2(CH2)3NH2
NH CH
Met
M
Methionine
CH2CH2SCH3
NH CH
Phe
F
Phenylalanine
CO
CO
H2C
HN CH CO
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Threeletter
One-letter
code
code
Pro
P
Name
Formula
Proline
N CH
Ser
S
Serine
H2C OH
NH CH
Thr
Trp
T
W
CO
Threonine
CO
CH(OH)CH3
NH CH
CO
H2C
NH
Triptophan
HN CH CO
Tyr
Y
CH3
Tyrosine
H2C
HN CH CO
Val
V
Valine
CH(CH3)2
NH CH
Ahx
*
6-aminohexanoic
acid
NH
(CH2)5
CO
CO
Table I Abbreviations used for amino acid residues according to the
Biochemistry Nomenclature Committee of the IUPAC-IUB [specified in Eur. J.
Biochem. 138, 9-37 (1984) and J. Biol. Chem. 264, 633-673 (1989)]. α carbon
side chains are presented in the non-ionic form for the twenty coded amino acids;
All amino-acid residues employed corresponded to the natural L-configuration.
* Ahx is a non-coded amino acid residue used in this work.
viii
Amino acid protecting groups
Abbreviation
Name
Stability
Formula
Boc
t-butyloxycarbonyl
Stable to bases,
labile to TFA
CH3
O
H3C C O
CH3
O
Fmoc
9-fluorenylmethyloxycarbonyl
Npys
3-nitro-2-pyridylsulphenyl
Stable to acids and
labile to bases
O
Stable to acids and
bases, labile to
nucleophiles
S
NO2
N
H3C
Pmc
2,2,5,7,8pentamethylchromane-6sulphonyl
Stable to bases,
labile to TFA
CH3
H3C
CH3
H3C
O S O
t
Bu
Trt
t-butyl
Triphenylmethyl (trityl)
Stable to bases,
labile to TFA
Stable to bases,
labile to 1% TFA
CH3
H3C C
CH3
C
Table II Amino acid protecting groups employed in this work.
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Resins, handles and coupling reagents
Abbreviation
Structure
OCH3 NH2
AM
H3CO
OH
O
O
MBHA
H2N
Polystyrene
PEG-PS
H2N
O
H
N
O
DIP
O
N
H
(
O)
n
H3C
H3C
H
N
N
H
O
CH N C N CH
O
N
H
CH3
CH3
CH3 CH3
H3C N
TBTU
+
N CH3
-
BF4
N
N
N+
O
-
Table III Resins, handles and coupling reagents used in this work.
x
Polystyrene