Biosensors to detect enzymeligand and

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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) Lecturer
Po-Huang Liang ???, Associate Research
Fellow Institute of Biological Chemistry,
Academia Sinica Tel [phone] ext. 6070
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Stopped-flow for measurements of protein-protein
and protein-small molecule interaction
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Substrate binding kinetics
k1S
Rate dE/dt -k1SE dE/E
-k1Sdt ln(Et / Eo) -k1St Et Eo
exp (-k1St) ES Eo-Et Eo(1-exp
(-k1St)) kobs k1 S
E ES
k1S
kobs k1S k-1 The slope of kobs vs S
gives kon and intercept gives koff
E ES
k-1
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3-D structure of E. coli UPPs
Two 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
The amino acids in a1 area are important for
catalysis and substrate binding D26 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 Triton bottom no Triton

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Long-lived intermediate C30 formed by A69L and
C35 by L67W
A69L
L67W
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Active site topography of UPPs
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Synthesis of FsPP to Probe UPPs Conformational
Change Chen et al.(2002) J. Biol. Chem. 277,
7369-7376.
FsPP
Ki of FsPP as an inhibitor 0.2 mM kcat of FsPP
as an alternative substrate 3 x 10-7 s-1
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Stopped-Flow experiments
UPPs-FPP IPP
UPPs-FsPP IPP
3 phases in 10 sec
1 phase in 0.2 sec
Binding rates vs. IPP gives IPP kon 2 mM-1
s-1
2 phases in 0.2 sec
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Different level of Trp fluorescence quench by FPP
wild-type
W31F has less quench
FPP binding mainly quenches the fluorescence of
W91, a residue in the a3 helix that moves toward
the active site during substrate binding
W91F has almost no quench
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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 is required fro IPP binding
Mg2
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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
Ko et al., (2001) J. Biol. Chem. 47474-47482
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Model of UPPs conformational change during
catalysis
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Fluorescent probe for ligand interaction and
inhibitor binding
Chen et al., (2002) J. Am. Chem. Soc. In press
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Synthesis of Fluorescent Substrate Analogue
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Characterization of the fluorescent probe
(A)
(B)
(A) Fluorescence is quenched by UPPs and
recovered by replacement with FPP (B) Probe binds
to UPPs with 11 stoichiometry
(C)
(D)
(C ) Probe binds to UPPs with a kon 75 mM-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?
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Reaction DHF NADPH THF NADP
Association
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Competition experiments to measure Dissociation
rate constants using Stopped-flow
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Rate constant for the pre-steady-state
burst measured 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 pH and 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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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
Association kinetics of ColE9-Im9 complex (lower)
and E9-DNase-Im9 (upper), ColE9 0.35 mM, Im
1.75 7 mM.
Dissociation kinetics of ColE9-Im9 complex at 0
and 200 mM NaCl. The preincubated E9 with 3HIm
(6 mM) was mixed with unlabeled Im (54 mM)
k2
k1
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
N I NI NI
k-1
K-2
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BIACORE (Biosensor)
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Sensor chip and coupling
CM5 couple ligand covalently
NTA bind His-tagged lignad SA 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 (ml/min) and immobilized
level (RU) for different experiments
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Definition
ka
A B AB
kd

• 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/AB
• ---KD kd/ka AB/AB
• ---range pm to uM

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Association and dissociation rate constant
measurements
ka
A B AB
kd
In solution at any time t At Ao AB
Bt Bo AB dAB/dt kaAtBt
kdABt In BIAcore at any time t At C AB
R Bo Rmax thus Bt Rmax R dR/dt
kaC(Rmax-Rt) kd (R)
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It is easy to mis-interpret the data
It
Distinguish between fast binding and bulk effect
use reference or double reference
Two ways to overcome mass transfer limitation
1.increase flow rate 2. 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)

• Phenyl-Sepharose
• Q-Sepharose

HEK affinity column
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Ion-exchange
RP-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-2 Identifies major
IGF binding site determination in both the N- and
C-terminal domains J. Biol. Chem. (2001) 276,
27120-27128.
IGFBPs contain Cys-rich N- and C-terminal and
a linker domains. The truncated bIGFBP-2
were generated and their interaction with IGF
were studied.
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Lane 2 1-279 IGFBP-2His Lane 3 1-132
IGFBP-2 Lane 4 1-185 IGFBP-2 Lane 5 96-279
IGFBP-2His Lane 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 DT1 (Ts Tr) negative. A cell
feed-back (CFB) supplies power on a heater on the
sample cell to drives the DT1 back to zero.
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Binding isotherms
Simulated isotherms for different c values c K
(binding constant) x macromolecule concentration
c should be between 1 and 1000 Make 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 (DH).
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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 the formation of the active
Ras-GTP.
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Example OBrien et al., Alternative modes of
binding of proteins with tandem SH2 domains
(2000) Protein Sci. 9, 570-579

• pY110/112 bisphosphopeptide
• binds to ZAP70 showing a 11 complex
• (B) Monophoshorylated pY740 binds
• to p85 with two binding events
• (C) Binding of pY740/751 peptide into
• p85. The asymmetry of the isotherm
• shows two distinct binding events
• showing that an initial 21 complex of
• protein to peptide is formed. As further
• peptide is titrated, a 11 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

• For AZP70, SH2 proteinpeptide 11
• For p85 (or NiC), initial titration results in
  peptide SH2 protein 0.51,
• adding more peptide to reach 11 complex.

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Interactions between SH2 domains and
tyrosine phosphorylated PDGFb receptor sequences
Panayotou et al., Molecular and Cellular Biology
(1993) 13, 3567-3576

• SH2 protein only binds to
• Phosphorylated Y751P peptide
• (B) The inclusion of competing
• peptide in the buffer yields first-order
• dissociation

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
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Thomas et al., (2001) Kinetic and thermodynamic
analysis of the interactions Of 23-residue
peptide with endotoxin. J. Biol. Chem. 276,
35701-35706.
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