Biosensors to detect enzyme-ligand and Protein-protein interactions hysical parameters in binding studies-principles, t nd instrumentation ethods to probe non-covalent macromolecular interac stopped-flow, BIAcore, and Microcalorimetry) cturer: Po-Huang Liang 梁梁梁 , Associate Research Fell stitute of Biological Chemistry, Academia Sinica l: 27855696 ext. 6070
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Biosensors to detect enzyme-ligand and Protein-protein interactions -Physical parameters in binding studies-principles, techniques and instrumentation.
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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
Stopped-flow for measurements of protein-protein and protein-small molecule interaction
(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
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
Reaction: DHF + NADPH THF + NADP+
Association:
Competition experiments to measureDissociation rate constants usingStopped-flow
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
Observed rate constants for hydride transfer as a function of pHand predictable kinetic behavior
Interaction of colicin E7 and immunity 7
Entrance of colicin E is through the BtuB (vitamin B12 receptor) and TolA
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)
•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
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
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)
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
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
Ion-exchangeRP-HPLC
The ligand is Al-1, which is previous found as ligand for EPH-like RTK family
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.
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.
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).
Signaling pathway of GPCR and RTK
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.
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.
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.
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
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.
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
Thomas et al., (2001) Kinetic and thermodynamic analysis of the interactionsOf 23-residue peptide with endotoxin. J. Biol. Chem. 276, 35701-35706.