biosensors to detect enzyme-ligand and protein-protein interactions
DESCRIPTION
Biosensors to detect enzyme-ligand and Protein-protein interactions . -Physical parameters in binding studies-principles, techniques and instrumentation -Methods to probe non-covalent macromolecular interaction (stopped-flow, BIAcore, and Microcalorimetry) - PowerPoint PPT PresentationTRANSCRIPT
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Biosensors to detect enzyme-ligand andProtein-protein interactions
-Physical parameters in binding studies-principles, techniques and instrumentation-Methods to probe non-covalent macromolecular interaction (stopped-flow, BIAcore, and Microcalorimetry)
Lecturer: Po-Huang Liang 梁博煌 , Associate Research FellowInstitute of Biological Chemistry, Academia SinicaTel: 27855696 ext. 6070
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Stopped-flow for measurements of protein-protein and protein-small molecule interaction
A B
Flow CellLight
Stop SyringeFluorescence Signal
Absorbance Signal
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Substrate binding kinetics
E ESk1[S] Rate = d[E]/dt = -k1[S][E]
d[E]/[E] = -k1[S]dtln([E]t / [E]o) = -k1[S]t[E]t = [E]o exp (-k1[S]t)[ES] = [E]o-[E]t = [E]o(1-exp (-k1[S]t))kobs = k1 [S]
E ESk1[S]
kobs = k1[S] + k-1
The slope of kobs vs [S] gives kon and intercept gives koff
k-1
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3-D structure of E. coli UPPsTwo conformers were found: one (closed form) with Triton bound and the other (open form) has empty active site
Ko, T. P. et al, (2001) J. Biol. Chem 276,47474-47482.
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Substrate-binding site
UPPs kcat (s-1) Km (FPP) (M) Km (IPP) (M) relkcat
a
wild type 2.5 0.1 0.4 4.1 0.3 1
akcat relative to that of wild type
¡Ó0.1¡Ó ¡Ó
49
3.0 27
1 x 10-4
¡Ó
¡Ó5
0.7 ¡Ó
0.7 ¡Ó0.1(2.4 0.1) x 10-4¡ÓR194A
R200A (2.5 0.2) x 10-3¡Ó 3 1 x 10-3
W31F 1.1¡Ó0.1 2.0 ¡Ó0.3 5.9 ¡Ó0.8 0.4
R30A 1.2 ¡Ó0.3 1108 ¡Ó250
0.4¡Ó0.1 1.6 ¡Ó0.3 65 ¡Ó10 0.2R39A
(3.0 0.3) x 10-2¡Ó 1 x 10-2
0.7 ¡Ó0.1 16 ¡Ó2N28A (3.0 0.3) x 10-2¡Ó 1 x 10-2
H43A (2.6 0.2) x 10-3¡Ó 3 ¡Ó0.3 63 ¡Ó3.6 1 x 10-3
14.1 1.40.5
0.7 280 20
1.3 x 10-3
1 x 10-2
(3.30 0.03) x 10-3
(2.60 0.02) x 10-2
D26A
E213A ¡Ó
¡Ó0.1
0.1
¡Ó
¡Ó
¡Ó
¡Ó
The amino acids in 1 area are important for catalysis and substrate bindingD26 is located in a P-loop conserved for pyrophosphate binding
Pan et al., (2000) Biochemistry 39, 13856-13861
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Large L137 on the bottom controls product chain length
upper: + Tritonbottom: no Triton
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Long-lived intermediate C30 formed by A69Land C35 by L67W
A69L
L67W
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Active site topography of UPPs
L137
A69
Flexible loop
32
1
O P O P O--O
-O
O O
-O
PO
O-
OP
O
O-
O
chain elongation
E213D26 Mg2+
Mg2+
L67BD
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Synthesis of FsPP to Probe UPPs Conformational Change Chen et al.(2002) J. Biol. Chem. 277, 7369-7376.
P
O
MeO OMeOMe
1 equiv Bu4NOHP
O
MeO O-
OMeP
O
MeO OOMe
POMe
S
OMe
5.64 equiv TMSI
24 h, 94%100 oC Acetonitrile
-35 oC, 30 min
1 equiv (OMe)2P(S)Cl
-35 oC rtover 6 h
30~35%
P
O
TMSO OOTMS
PSTMS
O
OTMS
P
O
-O OO-
PS-
O
O-
Bu4NOH/H2OP
O
-O OO-
PS
O
O-
0.45 equiv farnesyl chloride
Acetonitrile25 oC
6 h, 70%
3 NH4+
FsPP
Ki of FsPP as an inhibitor = 0.2 M kcat of FsPP as an alternative substrate = 3 x 10-7 s-1
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Stopped-Flow experimentsUPPs-FPP + IPP
UPPs-FsPP + IPP
Binding rates vs. [IPP] gives IPP kon = 2 M-1 s-1
3 phases in 10 sec
2 phases in 0.2 sec
1 phase in 0.2 sec
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300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500Fl
uore
scen
ce In
tens
ity (a
.u.)
Wavelength (nm)
300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500
Fluo
resc
ence
Inte
nsity
(a.u
.)
Wavelength (nm)
300 320 340 360 380 400 420 4400
500
1000
1500
2000
2500
3000
3500
Fluo
resc
ence
Inte
nsity
(a.u
.)
Wavelength (nm)
Different level of Trp fluorescence quench by FPP
wild-type W31F has less quench
W91F has almost no quench
FPP binding mainly quenches thefluorescence of W91, a residue inthe 3 helix that moves toward theactive site during substrate binding
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FPP binding does not require Mg2+; IPP binding needs Mg2+
FPP (or FsPP) quenches the UPPs Intrinsic fluorescence even in the absence of Mg2+
+ Mg2+
Mg2+ is required fro IPP binding
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The role of a flexible loop of residues 71-83
The invisible loop in the E. coli UPPs structure is responsible to bring IPP to the correct position and orientation to react with FPP
UPPs kcat (s-1) Km (FPP) (M) Km (IPP) (M) relkcat
a
wild type 2.5 0.1 0.4 4.1 0.3
133 14
16.2 2.2
1.0
0.4
8
1.6 15.7
1
0.04
0.1
0.01
0.30 0.01
S71A
E73A
N74A
akcat relative to that of wild type
¡Ó
¡Ó
¡Ó
¡Ó
¡Ó
0.1
0.2
0.1
0.6
0.3
¡Ó
¡Ó ¡Ó
¡Ó
¡Ó
¡Ó¡Ó
E81A 0.4 0.1¡Ó 0.4¡Ó0.1 88 10¡Ó 0.2
0.4 ¡Ó0.1(2.20 0.03) x 10-2¡Ó
R77A (1.4 0.1) x 10-4 5 x 10-5
2.5
0.11 0.01¡Ó
W75A 1.1¡Ó0.1 3.2 ¡Ó0.3 46 ¡Ó4 0.5
Ko et al., (2001) J. Biol. Chem. 47474-47482
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Model of UPPs conformational change during catalysis
Flexible loop
32
1
O P O P O--O
-O
O O
-O
PO
O-
OP
O
O-
OFlexible loop
32
1
Mg2+
closed-form open-form
Chain elongation
W75W31
binding release
L137
W91
E213 -O
P
O
O-
OP
O
O-
O
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OPP
PPOPPO
Inhibitor
Fluorescent probe for ligand interaction and inhibitor binding
Chen et al., (2002) J. Am. Chem. Soc. In press
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OH O O O OOH
O OBr
O OOO
R
O
O OHOO
R
O BrOO
R
O OOO
R
P O
OP O-
O
O-O-
1 2 3
4 5a R = CH35b R= CF3
(a) DHP, PPTs, CH2Cl2, 90%; (b) SeO2, t-BuOOH, salicylic acid, CH2Cl2, 32%; (c) NBS, Me2S, CH2Cl2, 92%; (d) 5a. K2CO3, DMF, 7-hydroxy-4,8-dimethylcoumarin, 98%, 5b. K2CO3, DMF,7-hydroxy-8-methyl-4-trifluoromethylcoumarin, 94%; (e) PPTs, EtOH, 6a. 99%, 6b. 92%; (f) CBr4, PPh3, CH2Cl2, 7a. 87%, 7b. 80%; (g) (n-Bu4N)3HP2O7, CH3CN, 8a. 54%, 8b. 46%.
a b
c d
e f
g
6a R = CH36b R= CF3
7a R = CH37b R= CF3
8a R = CH38b R= CF3
3 NH4+
Synthesis of Fluorescent Substrate Analogue
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400 450 500 550 6000
200
400
600
800
1000
1200
Fluo
resc
ence
Inte
nsity
(a.u
.)
Wavelength (nm)
0.0 0.5 1.0 1.5 2.0 2.5 3.0
200
300
400
500
600
700
Fluo
resc
ence
Inte
nsity
(a.u
.)
Concentration ()
Characterization of the fluorescent probe
(A) Fluorescence is quenched by UPPs and recovered by replacement with FPP(B) Probe binds to UPPs with 1:1 stoichiometry
(A) (B)
(C) (D)
(C ) Probe binds to UPPs with a kon = 75 M-1 s-1
(D) Probe releases from UPPs (chased by FPP) with a koff = 31 s-1
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Substrate and product release rate
FPP is released at 30 s-1 UPP is released at 0.5 s-1
Can this method apply to drug-targeted prenyltransferases to find non-competitive inhibitor?
IPPE + FPP E-FPP
fast
30 s-1E-FPP-IPP E-C20
E-C25 E-C30 E-C35 E-C40
E-C45 E-C50 E-C55 E + C55
2.5 s-1 2 s-1
3.5 s-1 2.5 s-1 3 s-1 3.5 s-1
3 s-13.5 s-1 0.5 s-1
2 M-1 s-1
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Reaction: DHF + NADPH THF + NADP+
Association:
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Competition experiments to measureDissociation rate constants usingStopped-flow
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Rate constant for the pre-steady-state burstmeasured by stopped-flow energy transfer
Uisng NADPH, 450 s-1 is followed by a 12 s-1 steady-state rate.Using NADPD, 150 s-1 is followed by the same rate at pH 6.5, isotope effect kH/kD =3
Pre-steady-state rate is decreased with pH
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Observed rate constants for hydride transfer as a function of pHand predictable kinetic behavior
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Interaction of colicin E7 and immunity 7
Entrance of colicin E is through the BtuB (vitamin B12 receptor) and TolA
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kon = 4 x 109 M-1 s-1
koff = 3.7 x 10-7 s-1
Kd = koff /kon = 7.2 x 10-17 M
Association kinetics of ColE9-Im9 complex (lower) and E9-DNase-Im9 (upper), [ColE9]
= 0.35 M, [Im] = 1.75 – 7 M.
Wallis et al., Protein-protein interactions in colicin E9 Dnase – immunity protein complexes. 1. Diffusion controlled association binding for the cognate complex Biochemistry 1995, 34, 13743-13750
Dissociation kinetics of ColE9-Im9 complex at 0 and 200 mM NaCl. The preincubated E9with [3H]Im (6 M) was mixed with unlabeled Im (54 M)
N + I NI* NIk1
k-1
k2
K-2
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BIACORE (Biosensor)
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Sensor chip and couplingCM5: couple ligand covalently
NTA: bind His-tagged lignadSA: capture biotinylated biomolecules
HPA: anchor membrane bound ligand
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SPR: surface plasmon resonance
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Objects of the experiments
•Yes/No binding, ligand fishing•Kinetic rate analysis ka, kd
•Equilibrium analysis, KA, KD
•Concentration analysis, active concentration, solution equilibrium, inhibition
Control of flow rate (l/min) and immobilized level (RU)for different experiments
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Definition
•Association rate constant: ka (M-1 s-1)---Range: 103 to 107
---called kon, k1
•Dissociation rate constant: kd (s-1)---Range: 10-5 to 10-2
---called koff, k-1
•Equilibrium constant: KA (M-1), KD (M)---KA = ka/kd = [AB]/[A][B]---KD = kd/ka = [A][B]/[AB]---range: pm to uM
A + B ABka
kd
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Association and dissociation rate constant measurements
A + B ABka
kd
In solution at any time t : [A]t = [A]o – [AB]; [B]t = [B]o – [AB]d[AB]/dt = ka[A]t[B]t – kd[AB]tIn BIAcore at any time t: [A]t = C; [AB] = R; [B]o = Rmax thus [B]t = Rmax – Rd[R]/dt = ka*C*(Rmax-Rt) – kd (R)
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It
It is easy to mis-interpret the data
Distinguish between fast bindingand bulk effect: use referenceor double reference
Two ways to overcome mass transfer limitation: 1.increase flow rate2. reduce ligand density
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Example 2: Lackmann et al., (1996) Purification of a ligand for the EPH-like receptor using a biosensor-based affinity detection approach. PNAS 93, 2523 (ligand fishing)
HEK affinity column
(A) Phenyl-Sepharose(B) Q-Sepharose
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Ion-exchangeRP-HPLC
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The ligand is Al-1, which is previous found as ligand for EPH-like RTK family
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BIAcore analysis of bovine Insulin-like Growth Factor (IGF)-binding protein-2Identifies major IGF binding site determination in both the N- and C-terminal domainsJ. Biol. Chem. (2001) 276, 27120-27128.
IGFBPs contain Cys-rich N- and C-terminal and alinker domains. The truncated bIGFBP-2 weregenerated and their interaction with IGF werestudied.
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Lane 2: 1-279 IGFBP-2HisLane 3: 1-132 IGFBP-2Lane 4: 1-185 IGFBP-2Lane 5: 96-279 IGFBP-2HisLane 6: 136-279 IGFBP-2His
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MicroCalorimetry System Right: ITC (Isotheromal titration Calorimetry)
Inject “ligand” into “macromolecule”
A small constant power is applied to the reference To make T1 (Ts – Tr) negative. A cell feed-back(CFB) supplies power on a heater on the sample cell to drives the T1 back to zero.
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Binding isotherms
Simulated isotherms for different c valuesc = K (binding constant) x macromoleculeconcentrationc should be between 1 and 1000Make 10-20 injections
can be used to obtain binding affinity or binding equilibrium constant (Keq),molecular ration or binding stoichiometry (n),And heat or enthalpy (H).
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Signaling pathway of GPCR and RTK
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Activation of Ras following binding of a hormone (e.g. EGF)to an RTK
GRB2 binds to a specific phosphotyrosine on the activated RTK and to Sos, which in turn reacts with inactive Ras-GDP. The GEF activity of Sos then promotes theformation of the active Ras-GTP.
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Example: O’Brien et al., Alternative modes of binding of proteins with tandem SH2 domains (2000) Protein Sci. 9, 570-579
(A) pY110/112 bisphosphopeptide binds to ZAP70 showing a 1:1 complex
(B) Monophoshorylated pY740 binds to p85 with two binding events
(C) Binding of pY740/751 peptide intop85. The asymmetry of the isotherm shows two distinct binding eventsshowing that an initial 2:1 complex of protein to peptide is formed. As further peptide is titrated, a 1:1 complex is formed.
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ITC data for the binding of peptides to ZAP70, p85, NiC, and isolated c-SH2 domain
KB1 and KB2 correspond to the equilibrium binding constants for the first and the second binding events.
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Conformational change of two SH2 binding with phosphorylated peptide
(A) Primary sequence NiC(B) a. NiC; b.NiC + bisphosphorylated peptide (C ) a. N-terminal SH2 alone; b.N-terminal SH2 + pY751 peptide; c. C-terminal SH2; .d. C-terminal SH2 + pY751 peptide
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Model for binding of bisphosphorylated peptide to the SH2 domain
(A) For AZP70, SH2 protein:peptide = 1:1(B) For p85 (or NiC), initial titration results in peptide: SH2 protein = 0.5:1, adding more peptide to reach 1:1 complex.
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Interactions between SH2 domains and tyrosinephosphorylated PDGF – receptor sequences
(A) SH2 protein only binds to Phosphorylated Y751P peptide(B) The inclusion of competing peptide in the buffer yields first-orderdissociation
The N-terminal SH2 domain bound with high affinity to the Y751P peptide but not to the Y740P, whereas C-terminal SH2 interacts strongly with both
Panayotou et al., Molecular and Cellular Biology (1993) 13, 3567-3576
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Thomas et al., (2001) Kinetic and thermodynamic analysis of the interactionsOf 23-residue peptide with endotoxin. J. Biol. Chem. 276, 35701-35706.
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