identification, production and purification - tap tag/ms foot-printing - single gene expression and...
TRANSCRIPT
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Identification, production and purificationIdentification, production and purification
- TAP tag/MS foot-printing- Single gene expression and in vitro assembly:- Co-expression techniques in E. coli and in insect cells - Purification strategies
Analysis of assembly processesAnalysis of assembly processes
- fluorescence methods- fluorescence methods- microcalorimetry- microcalorimetry- surface plasmon resonnance- surface plasmon resonnance
- NMR- NMR- equilibrium binding/allostery- equilibrium binding/allostery
Size and shapeSize and shape
- Dynamic Light scattering- SAXS and SANS- Analytical ultracentrifugation- non denaturing MS- Electron microscopy
- thermodynamics: the essentials
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Characterization Strategies for Characterization Strategies for non-obligatory complexesnon-obligatory complexes
Isolated components are available and pure/homogenous
Association ?(biochemical analysis)
Pull downNative gel eletrophoresis, EMSAGel filtration
AUC, SAXS, SANSNative MS, EM
Thermodynamic parameters ? EMSA, ITC, spectroscopy, AUC, SPR…..
Conformational homogeneity ? EM, SAXS, SANS, AUC, DSC…..
Association ?(biophysical analysis )
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Biochemical methods to detect associationBiochemical methods to detect association
Native gels for protein-protein and protein-NA complexes
- association of NR LDB and co-activator peptide
- EMSA, filter binding assays
- cross-link
-[DNA] = const.-[protein] varies
Kd by fitting to binding isotherm
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Biophysical methods to detect and characterize Biophysical methods to detect and characterize associationassociation
Thermodynamic parameters ? EMSA, ITC, spectroscopy, AUC, SPR…..
Structure ? EM, SAXS, SANS, AUC, DSC, MS…..
Fluorescence
Calorimetry
Surface Plasmon resonnance
NMR (Bruno Kieffer, march 31th)
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Isolated compounds availible (non obligatory complexes)
Signal that is different for the free and associated compounds
Principle
Titration
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Spectroscopic methods
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The electromagnetic spectrum
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Excitation(Absorption)
Emission(fluorescence)
h > h
<
Fluorescence
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Applications in biology
• Intensité de fluorescence et temps de vie sont très sensibles à l'environnement (100x) => sondes
• Molécules inhibitrices (quenching) => sonde d'accessibilité
• temps de vie de l'état excité (10-9 s = 1 ns), permet de mesurer des mouvements (absorption 10-15 s => molécule immobile)
• techniques de polarisation dépolarisation
• Transfert d'excitation (FRET) => mesure de distances entre chromophores
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Natural fluorophoresburied Tryptophane
amino acids: W, Y, F
Dyes
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• Protéine de 283 aa issue d'une méduse (Aequorea victoria), naturellement fluorescente.
• Chromophore : modification d'une séquence GYS • lexc = 395 nm, 475 nm ; lemis = 504 nm
GFP (green fluoresent protein)
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Split proteins for in vivo detection of protein-protein interactions
Split GFP as a probe for detecting protein-protein interactions. Protein A and protein B are linked to the split VDE intein. Interaction between protein A and protein B induces the folding of N- and C-terminal halves of VDE and protein splicing occurs. The N- and C-terminal halves of GFP are linked together by a peptide bond to yield fluorescent GFP.
Copyright (c) 2006, Ozawa lab.
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The emission spectrum depends on the environment of the fluorophore
Thermofluor: follow denaturation of proteins and stabilization uponligand/DNA/peptide binding
buried Tryptophane
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Fluorescence anisotropy
one molecule (DNA, peptide or protein) is fused to a fluorophore
measure static fluorescence anisotropy
association results in an entity which rotates more slowly than the isolatedfluorophore and results in less scrambling of polarized light
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Fluorescence Polarisation
Fluorescence is measured with a linearly polarized beam
Vertical emission
Horizontal emission
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Rapid rotational diffusion
Intensities of vertical and horizontal emission almost equal
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Slow rotational diffusion
Intensities of vertical and horizontal emission differ = Anisotropy
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Polarisation de fluorescenceLoi de Perrin
• L'anisotropie de fluorescence est définie comme :
• En considérant que la probabilité d'excitation est proportionnelle à cos2(q), on peut montrer que la valeur maximum possible de l'anisotropie est :
• Dans la réalité, les valeurs de A0 sont inférieures, et :
AF// F
FF// F
F// 2F
A0max
2
5
AA0
1 F c
F
c
temps de vie de fluorescence
temps de correlation de rotation
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HATHAT
CoACoA
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Peptide co-activator binding
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• Un fluorophore à l'état excité (D) peut transmettre son énergie à un autre (A) par transfert non-radiatif, à distance.
– La fluorescence de D est partiellement éteinte tandis que celle de A apparaît.
– Cet effet résulte d'une interaction entre les dipôles électriques de D et A.
• Conditions pour le transfert
– La distance DA doit être relativement courte (effet en 1/r6)
– Recouvrement des bandes d'émission de D et d'absorption de A.
Fluorescence Resonnace Energy Transfer
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Transfert de fluorescence
• On définit une constante de vitesse de transfert kT
– r0 : distance de Förster, caractéristique du couple DA (distance pour laquelle on observe une efficacité de transfert de 50%)
– tf : temps de vie du donneur en l'absence de l'accepteur
• L'efficacité de transfert est donné par :
• La distance DA peut être calculée :
1
T
1
f kT
E kT
f
1 kT
r06
r06 r6
kT 1
f
r0r
6
T
f
T
f
1 E
rr01 EE
1/6
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• In vivo - cells co-transfected with plasmids expressing CFP and YFP fusion proteins- interaction between A and B and its modulation by effectors
CFP-A + YFP-B
CFP-AYFP-B
410 nm
In vivo and in vitro FRET analysis
• In vitro- HTP screening for compounds that inhibit A/B interaction
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Micro Calorimetry
La manière la plus directe de mesurer les paramètres thermodynamiques:étude des échanges de chaleur G, H, S et Cp
Energies mises en jeu faibles lors par exemple de processus d’association
Kd G°10 –3 -17 kJ/mol
10 –6 -34 kJ/mol
10 – 9 -51 kJ/mol
10 –12 -68 kJ/mol
Utilisation de la calorimétrie longtemps limitée par le manque de précisiondes calorimètresDéveloppements technologiques ont permis la production de calorimètressensibles :seuils de détection de l’ordre de la cal/sec
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Isothermal Titration Calorimetry (ITC)
Technique permettant l’étude de processus résultantde l’addition d’un composant dans la cellule de mesure. (eg titration d’une protéine par un ligand)
Mesure les enthalpies de réaction:
Anatomie d’un microcalorimètre:- cellule contenant la macromolécule T=k- seringue contenant le ligand.
Lorsque le ligand et la macromolécule interagissent, de la chaleur est absorbée ou dégagée selon que la réaction est endo ou exo thermique. Le système de régulation du calorimètre injecte ou absorbe de la chaleur pour maintenir la température constante.
Le signal mesuré est une puissance électrique.
Au cours d’une expérience ITC, un "ligand" est titré dans une
cellule contenant une solution de "macromolécule".
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A intervalles de temps réguliers, du ligandest injecté dans la cellule de mesure.
Les données brutes sont constituées par lacourbe puissance échangée = f(t). Cette courbe est constituée par une série de pics (chaque pic correspondant à une injection). L’aire de chaque pic représente la chaleur totale libérée pour l’injection.
Lorsque les sites de fixation du ligand sont saturés le signal diminue jusqu’à une valeur correspondant à la chaleur de dilution
Les données brutes permettent d’accéder à une isotherme de titration: chaleur échangée = f(rapport molaire ligand/macromolécule).
Une expérience ITC
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Fixation des sous unités E1 et E3 au PSBD core domain
Protein Science (2002),11 :1091-1100.
PSBD 180-230 ME1 7.5 ME3 13.5 M
H°n
Kd
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Differential Scanning Calorimetry (DSC)
Dédié à l’étude de la stabilité thermique des molécules
Equipé d’un système de régulation permettant la mesure des échanges de chaleur (Q produite ou absorbée) entre les cuves « échantillons » et « référence » (contenant uniquement le tampon).
Mesure l’excès de capacité calorifique présent dans la cuve échantillonCp (mcal/mol/degré) = f(T).
Dénaturation thermique des macromolécules: Tm, Cp, H dénaturation
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Molécule d’intérêt est dans la cuve de mesure.
Dans la cuve de référence le tampon dans lequel se trouve la macromolécule.
Au cours d’une expérience on fait varier la température et on mesure l’énergie
à fournir à la cuve de mesure pour que cuves de mesures et de référence soit
exactement à la même température. On mesure l’excès de capacité calorifique présent dans la cuve échantillon: Cp = f(T)
Référence Echantillon
T1
Q
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As the protein is heated, it reaches a temperature at which a large amount of heat is suddenly absorbed,as the protein unfolds.
The area under the curve represents the heat absorbed on denaturation. The temperature at the midpoint is the Tm of the protein. (Why would the Tm be dependent on the pH of the solution?)
There are two Cp's evident.
Cd is associated with the actual denaturation process and is analogous to the change in heat capacity observed in phase changes, such as solid to liquid water.
Cp which is the difference in heat capacity between the denaturated and native state. As was the case for the transfer of benzene to water, the Cp for protein denaturation is also positive, suggesting that in protein denaturation, hydrophobes are transferred from the interior of the protein to water.
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Cp = dH/dT= TdS/dT. (derived from from Maxwell's relationships).
Cp = d(H)/dT = Td(S)/dT
A positive Cp occurs when H and S are dependent on temperature, which is observed when a hydrophobe is transfer from a more nonpolar environment to water.
A negative Cp is observed when hydrophobes in water are transferred to a more nonpolar environment: formation of a protein-protein or protein-NA interface
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Formulation stability studies
Macromolecule domain structure
Chemical half-life determinations
Determining thermal stability and reversibility
Applications of DSC
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Thermodynamics and structure
IMFs: electrostatics, H-bonds, V der Waals, hydrophobic effect
les acides aminés hydrophobes pointent vers l’intérieur de la molécule
les paramètres thermodynamiques peuvent être estimés à partir de
la surface d’interaction (Cp).
Comment sa contribution est elle étudiée?
- théoriquement en mesurant
la surface accessible au solvant
(25 cal/mol/Å2)
- expérimentalement en mesurant des
différences de stabilité entre mutants ponctuels.
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Dans le cadre de relations structure-fonction, l’utilisation
de mutants ponctuels permet d’évaluer quantitativement les
contributions énergétiques des diverses interactions.
Pour une interaction protéine/ligand, on mesure les Kd pour
la protéine wt et différents mutants (qui n’affectent que l’interaction
Étudiée). On en déduit la contribution des diverses interactions à la formation du complexe.
Effets thermodynamiques des mutations
Er complexé à l’oestradiol
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wt: 3 interactions mt: 2 interactions
P(wt) + L P(wt)-L
P(mt) + L P(mt)-L
rG° = rG° (mt) – rG° (wt)avecrG‡ = -RTln ([P-L]/[P][L])
rG° (wt)
rG° (mt) G°#0G°#0
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Application à la Tyrosyl-tRNA synthétase
Structure de l’intermédiaire réactionnel [TyrRS-tyrosyl-adénylate]
Modèle de l’état de transition
Mute les résidus impliqués dans lareconnaissance et la stabilisation de l’état de transition
Mesure les paramètres cinétique del’enzyme wt et des mutants (kcat Km)
On en déduit les contributions énergétiques G‡ = -RTln (kcat/KM) des chaînes latérales des différents acides aminés à la stabilisation de l’état de transition.
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Théorie de l’état de transition
Enzyme = Catalyseur.
Une réaction doit être thermodynamiquement possible
Il faut que la vitesse de réaction ne soit pas nulle.
A + B X ‡ P + Q K‡ k’
v = dP/dt = k’ [X ‡]
X ‡ : complexe activé en équilibre rapide avec A et B (X = A, B , E)
K ‡ = [X ‡]/AB
- RT ln [X ‡] = G‡
v = k’ [A][B] exp(-G‡/RT)
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G‡ : différence d’énergie libre entre A, B et X ‡ Rôle de l’enzyme: diminuer G‡ , ie la hauteur de la barrière d’activation
G
A+B (+Enz)
P+Q (+Enz)
X ‡
G‡
G
A + B X ‡ P + Q
v = k’ [A][B] exp(-G‡/RT)
Energie d’activation
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On compare G‡ pour différents mutants
Interprétation de l’Energie d’activation
G‡ représente la variation d’énergie libre standard associée à la formation
du complexe activé, c’est à dire à l’état de transition.
G‡ peut être estimée à partir des paramètres cinétiques
G‡ = G‡ (mt) – G‡ (wt)
G‡ = -RTln (kcat/KM)
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mt wt S
Phe-34 Tyr-34 Tyr 0.52
Gly-35 Cys-35 ATP 1.14
Ala-51 Cys-51 ATP 0.47
Gly-48 Asn-48 ATP 0.77
Gly-48 His-48 ATP 0.96
Ser-35 Cys-35 ATP 1.18
Phe-169 Tyr-169 Tyr 3.72
Gy-195 Gln-195 Tyr 4.49
Gly-35 Ser-35 ATP -0.04
Ala-51 Thr-51 ATP -0.44
G‡ = G‡ (mt) – G‡ (wt)(kcal/mol)
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Etudes de Stabilité
Les protéines existent généralement dans deux états:
Etat natif: (N) - conformation (généralement) unique- chaîne repliée- protéine active biologiquement
Etat dénaturé: (D)- chaîne dépliée- caractéristiques de pelotes statistiques- protéine inactive
N D
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Température: Les liaisons, de Van der Waals et électrostatiques sont déstabiliséesà haute température (énergies de liaison exothermiques).
pH: Généralement ionisation des chaînes latérales enfouies à des pH extrèmes. A pH élevé His, Tyr seront chargées négativement et auront donc tendance à pointer vers le solvant et donc déstabiliser la protéine.
Constante di-électrique: affecte les interactions électrostatiques.
Les paramètres qui gouvernent la stabilité des macromolécules
Des additifs comme des sels ou des sucres (sucrose, glucose, glycerol....) peuvent stabiliser des protéines (conservation et conditionnement des protéines)
T
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Pour comprendre comment les structures tertiaires des macromolécules (protéines isolées ou complexes) il faut être capable de mesurer l’énergie libre de dénaturation (GH20 ou Ke).
A 25 ou 37 °C, les macromolécules sont en exclusivement sous forme Native et les constantes d’équilibre sont souvent difficilement mesurables.
En déplaçant l’équilibre dans le sens de la dénaturation (pH extrêmes, agents chaotropes, températures) on accès expérimentalement à la constante d’équilibre ou une énergie libre dans d’autres conditions expérimentales. Puis, à l’aide d’un cycle thermodynamique, on calcule GH20 ou Ke.
Agents chaotropes: Urée, Guanidinium
Etudes de Stabilité
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Systèmes modèles: petites protéines ou complexes de structures
connues et pour lesquelles le processus de dénaturation
est réversible (RNAses, Barnase, Lysozyme, Tryp/DNA....)
Il faut disposer d’une observable qui permet de suivrele processus de dénaturation en fonction par exemple de la concentration en agent chaotrope
L’observable peut être: - activité enzymatique- signal de fluorescence, CD- viscosité- déplacement chimique...
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fN et fD fractions de protéine Native et Dénaturéey observable permettant de suivre la réaction
On exprime fD , fN en fonction de y
fN yN + fD yD = y fD + fN = 1 d’ou fD = (y - yN) / (yD - yN)
Dans un système à deux états, la courbe de dénaturation fD = fonction ([dénaturant]) est une sigmoîde (coopérativité folding)
On en tire Keq = [D]eq/[N]eq = fD/fN et Go = -RTlnKeq = -RTln[ fD/(1 - fD)]pour chaque concentration en dénaturant.
N D
Interprétation de la courbe de dénaturation
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A partir des données de dénaturation, on peut calculer l’énergie libre standard de dénaturation pour chaque concentration en agent chaotrope.
N(H20) D(H2O)
N(Urée) D(Urée)
GH20
G
Gt°(N) Gt°(D)
G = - Gt°(N) + GH20 + Gt°(D) = GH20 + Gt°(D) - Gt°(N)= GH20 + Gt° = a –m* [Urée]
En extrapolant à une concentration en urée nulle (et Gt° = 0)
observation expérimentale
GH20 = a
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Exemple de la protéine Arc: étude des déterminants de stabilité dans le cas d’une protéine dimérique
Mutations ponctuelles
Dichroisme circulaire
N2 2U
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Etude de l’enthalpie de dénaturation
rH°(T) = rH°(To)+ cp(T-To)
cp lié à la variation de la surface accessible au solvant.
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Binding kinetics
• Since biological systems are not at equilibrium the rate of binding and dissociation is critical
• For a simple 1:1 interaction (A + B AB)
• Rate of dissociation – dAB/dt = k diss[AB] – where kdiss is the dissociation rate constant (koff)
• Rate of association – dAB/dt = kass[A][B]– where kass is the association rate constant (kon)
• At equilibrium: dAB/dt = kass[A][B] - kdiss[AB] = 0 • kdiss[AB] = kass[A][B]
– kdiss/kass = [A][B]/[AB] – Since KD = [A][B]/[AB] it follows that
– kdiss/kass = KD
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Dissociation
• Any reaction of the form dAB/dt ∞ [AB] will be exponential so– i.e. [AB]t = [AB]oe-kdisst
– kdiss determined directly by curve fitting• The half life (t1/2) can be calculate as follows• Since at the t = t1/2
[AB]t/[AB]o = 0.5 = e-kdisst1/2
• It follows that -kdisst1/2= ln 0.5 = 0.693
• Thust1/2 = 0.693/koff
Dissociation ofA from BSymbols are data, lines are fitted curves
t1/2
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Association
• In most experimental system it is impossible to follow association alone in the absence of simultaneous dissociation
• For the simple interaction A + B AB
• d[AB]/dt = kass[A][B] – kdiss[AB]
• It can be shown that [AB]t = [AB]final (1- e-kobst)
where kobs = kass[A]+koff
• Thus one needs to know the koff and the [A] to calculate the kon
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Summary of affinity and kinetic constants biological interactions
Interaction kon (M-1s-1) koff (s-1) KD (M)
Cell-cell recognition molecules
105 1-10 10-5-10-4
Antibody/antigen 105 10-3 10-8
Cytokine/receptor 105 10-4 10-9
Enzyme/inhibitor (eg barnase/barnstar)
108 10-3 10-11
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Factors affecting kinetics
• The association rate constant does not vary that much– Association requires two proteins to collide in the correct
orientation and in the correct conformation– This will be similar for most proteins– The basic rate is about 105 M-1.s-1
– The rate can be accelerated by long range electrostatic forces
• Increased rate of collision• Steer binding sites into correct orientation• E.g. barnase/barnstar interaction
• The dissociation rate constant varies considerable and is responsible for most of the change in affinity constants– It is determined by the number and strength of bonds in
the contact interface– Depends on size of interface and the degree of surface-
shape and electrostatic complementarity
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• The BIAcore uses an optical method (surface plasmon resonance) to measure changes in refractive index.
• Macromolecules binding to a sensor surface leads to an increase in refractive index near the surface.
Surface plasmon resonance
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A BIAcore sensorgram
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Sensorchips
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FIN