Surface plasmon
resonance
S2004
Methods for characterization of biomolecular
interactions – classical versus modern
Mgr. Josef Houser, Ph.D.
houser@mail.muni.cz
Surface plasmon resonance (SPR)
(Rezonance povrchového plasmonu) – collective oscillation of
free electrons on metal-dielectric interface
History
1950’s1900’s
Anomalous light
reflection on metal
grating
(R. W. Woods)
Definition of plasmon
1980
First trials to use SPR in
biomolecular
interaction analysis
1990
First commercial
instrument (Biacore)
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Papers dedicated to SPR (PubMed)
SPR – Basic principles
At the conditions of total internal
reflexion (angle, wavelength) the
incoming beam evokes exponential
wave spread in opticaly less dense
environment.
At certain combination of incident angle and wavelength the free
electrons on the metal surface are excited, what causes decrease
in reflected light intensity.
This effect depends on refractive index that
varies with the analyte binding to the surfacebound
ligand.
SPR – Basic principles
Refractive index change = change in
light intensity at certain wavelength.
Corresponds also to change of mass
on the chip surface = protein/ligand
binding. (1 RU ~ 1 pg/mm2)
Correlation between SPR
Response and Surface
Concentration
SPR – Basic principles
One binding partner immobilized on the chip
surface (ligand), second is free in solution (analyte).
Association Dissociation
Simple binding - kinetics
• Typical binding curve – association and dissociation
phase, (surface regeneration)
1 Association
2 Dissociation
3 Regeneration1
2
3 3
Time s
Response
RU
Binding experiment – steady state
v(association) = ka * [analyte](solution)
v(dissociation) = kd * [analyte](bound)
[analyte](solution) >> [analyte](bound)
v(association) >> v(dissociation ) association phase
v(association) = v(dissociation) steady state
-> response is proportional to KD and Rmax
[analyte](solution) << [analyte](bound)
v(association) << v(dissociation ) dissociation phase
[ ][ ]
[ ]MX
XM
K
1
K
A
D ===
a
d
k
k
Receptor ligand interaction
[ ] [ ][ ] [ ]MXXM
MX
da kk
dt
d
-=
[ ] 0
MX
: =
dt
d
mequilibriu
+
• Kinetics of interaction
• Steady state
Surface plasmon
resonance
(SPR)
Same affinity but different kinetics
1035
• All 4 compounds have the same affinity KD = 10 nM = 10-8 M
• The binding kinetic constants vary by 4 orders of magnitude
1035kon koff
M-1s-1 s-1
106 10-2
105 10-3
104 10-4
103 10-5
Time Time
Response
Concentration = 1000 nM
Completely
blocked
target - all
target sites
occupied
Compounds with
slow off-rates
occupy the target
for a longer time
Concentration = 100 nM
Same affinity but different kinetics
koff (s-1)
kon(M-1s-1)
104
0.0001 0.001 0.01 0.1 1
107
106
105
102
103
1 nM100 pM 10 nM10 pM
100 nM
1 mM
1 mM
100 mM
10 mM
KD
HIV-p inhibitors: on-off rate map
Kinetics vs. affinity in Drug design
High affinity – first aim in drug discovery
BUT
May be caused by high ka and kd = fast dissociation (!)
Kinetics – lower ka AND kd may mean longer effect
This fact is known but usually not considered !
Experimental results
Simple binding - kinetics
• Typical binding curve – association and dissociation
phase, (surface regeneration)
1 Association
2 Dissociation
3 Regeneration1
2
3 3
Time s
Response
RU
Chip surface
Gold
Matrix
Protein
Analyte
Concentration
Analyte 1
Analyte 2
Specificity
0
100
200
300
400
500
0 100 200 300 400 500 600 700
RU
Response
sTime
0
100
200
300
400
500
0 100 200 300 400 500 600 700
time [s]
RU
0
100
200
300
0 200 400 600 800
time [s]
RU
concentration
concentration
Ligand 1
Ligand 2
Ligand 3
Fast complex association and dissociation
Fast equilibrium ð KA, KD
Slow complex association and dissociation
Kinetic constants ka, kd ð KA, KD
specificity
Simple binding – steady state
Fast association and dissociation data
are not easy to fit
BUT
v(association) = v(dissociation) steady state
-> response is proportional to KD and Rmax
-20
-10
0
10
20
30
40
50
100 150 200 250 300 350 400 450 500
RU
sTime
Response
Differential curves
Fast association and dissociation
KA, KD
RU
Response
Saccharide concentration
RUmax
½ RUmax
[Protein] [Analyte]
[Protein-Analyte]
KD
=
RU = ½ RUmax
[Protein] = [Protein-Analyte]
KD = [Analyte]
Direct binding assay
Factors influencing binding and
response
• Density of the molecules on chip
• Concentration of molecules in solution
• Strength of interaction between both molecules
• Total mass of interacting partner
• Portion of active molecules present –
proper sample characterization needed, changes upon
immobilization – site accessibility restriction,
conformational changes, intermolecular distance
Which binding partner to immobilize?
• Stability
• Availability
• Molecular mass
• Immobilization technique
• Multivalency
SPR Chip – rough scheme
Chip surface
Matrix
Protein/Ligand
Glass
Linker layer
Gold
Dextran layer
Specific layer
User-defined biospecific surface
• Biocompatible
• Low non-specific
binding
• Robust
• More than 100
runs on the same
surface
Various immobilization techniques
• Direct: covalent coupling
• Amine
• Thiol
• Aldehyde
• Carboxyl
• Capture
• Streptavidin - Biotin
• NTA-Ni2+-His
• Anti-his-His
• RaM Fc - MAb
• Anti-GST- GST
High flexibility in creating biospecific surfaces
Protein immobilization
Chip surface
Gold
Matrix
Protein (lectin)
Saccharide
N-ethyl-N’-(3-diethylaminopropyl)karbodiimide
N-hydroxysuccinimide
„Amine-coupling“
CM5 chip – surface modified by
carboxymethylated dextran
Chip surface
Matrix
Streptavidine
Biotin
Saccharide
Polyacrylamide
Typical spacer for saccharides is
-(CH2)3-, for biotin -(CH2)6Saccharide
immobilization
N-ethyl-N’-(3-diethylaminopropyl)karbodiimide
N-hydroxysuccinimide
„Amine-coupling“
Ni-NTA utilization
Activation
Protein binding
Regeneration
+Ni2+
Sample
application
+EDTA
Flexibility in Assay Design
Multiple assay formats providing complementary data
Direct measurement Indirect measurement
Inhibition in solution assay (ISA)
Surface competition assay (SCA)Direct Binding Assay (DBA)
Time
Response
Inhibitor
concentration
Response0 %
50 %
100 %
Chip surface
Gold
Matrix
Immobilized
saccharide
Lectin
Free saccharide
Inhibition in solution assay
Inhibitor
concentration
Response
-200
-100
0
100
200
300
400
500
600
700
800
0 100 200 300 400 500
Time s
Rel.response
RU
0-250 μM D-mannose
Inhibition of binding of
B. cenocepacia lectin
to immobilized D-mannose
Inhibition in solution assay
Inhibitor
concentration
Response
0-80 mM D-galactose
-200
-100
0
100
200
300
400
500
600
700
800
0 100 200 300 400 500
Time s
Rel.response
RU
Inhibition in solution assay
Inhibition of binding of
B. cenocepacia lectin
to immobilized D-mannose
32
IC50D-mannose
IC50saccharide
Effectivity=
Lectin from B. cenocepacia:
Benzyl-α-D-mannoside ≈ Methyl-α-D-mannoside ≈
D-mannose » L-fucose > D-arabinose > L-galactose >
Methyl-α-L-fucoside » D-galactose
0 %
50 %
100 %
Inhibition in solution assay
Two channels necessary - reference
• “Non-interacting” surface serves as a blank
• Elimination of non-specific interactions
• Enhancement of weak interaction resolution
• Possible reference surfaces:
• Unmodified surface – gold, dextran layer,…
• Activated and blocked surface without immobilized
ligand/protein
• Inactivated/non-functional protein
-50
0
50
100
150
200
250
-50 0 50 100 150 200 250 300
Time s
Response
RU
Two channels necessary - reference
Reference channel
Multichannel set-up
• One or more references
• Multiple channels – 2, 4, 6, 36,…
• Multiple detection spots
ØHigh throughput
ØParallel reference
Specialized techniques
• Membrane proteins
• Multi-layer approaches – antibodies, protein
complexes
• Whole cell immobilization
• Thermodynamics measured by SPR
• Ligand recovery
On-surface reconstitution approach
ü A very quick and easy method for functional reconstitution
of immobilized membrane proteins with lipids.
ü Conventional immobilization techniques are applicable on
membrane proteins.
ü Surfaces with high density of receptor can be prepared.
ü The lipid matrix can be renewed after every cycle.
ü “Lipid bilayers” can be very rapidly and easily built and
rebuilt on Pioneer Chip L1 (Biacore).
Immobilize a GPCR-specific mAb on a L1 chip.
Capture a detergent-solublized GPCR on the
immobilized mAb surface.
Reconstitute a lipid bilayer around
the receptor
Inject lipid/ detergent mixed micelles across the surface.
Wash the surface with buffer
Dissociates the detergent from the micelles.
Establish the integrity of the reconstituted GPCR
Use conformationally sensitive anti-bodies.
Study the kinetics of ligand/receptor interactions
Binding of the chemokine SDF1a
to the reconstituted CXCR4 receptor
-5
0
5
10
15
20
25
30
35
40
45
-10 0 10 20 30 40 50 60 70 80 90 100 110 120
Time (s)
Response(RU)
Time (s)
Response(RU)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
[SDF1a] (M)
Response(RU)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
[SDF1a] (M)
Response(RU)-5
0
5
10
15
20
25
30
35
40
45
-10 0 10 20 30 40 50 60 70 80 90 100 110 120
Time (s)
Response(RU)
Time (s)
Response(RU)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
[SDF1a] (M)
Response(RU)
-5
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
1.0E-09 1.0E-08 1.0E-07 1.0E-06 1.0E-05
[SDF1a] (M)
Response(RU)
KD = 288 ± 11 nM
Proteomics Study
BIACORE
“Classical Applications”Kinetics, Affinity
Structure-Activity studies
Receptor
Express 6-24 months
Recovery
Enzymatic
digestion
HPLC
separation
MS
Ligand
Fishing SPR/MS
Main SPR biosensors
• GE Healtcare – Biacore T200,
Biacore 4000, Biacore 3000, etc.
• Reichert – SR7000DC
• BioRad – ProteOn™ XPR36
• Biosensing Instrument – Bi4000,
Bi3000, etc. Biacore T200
SR7000DC
ProteOn™ XPR36 Bi4000
Biacore 3000 (GE Healthcare)
Simultaneous 4-channel system
Study of small molecules (200 Da),
proteins, complex mixtures, lipids,
viruses, prokaryotic and eukaryotic cells
Possibility to isolate binding partners for
subsequent MS analysis
Objectives of the SPR experiment
o Kinetic Rate Analysis: How FAST?
• ka, kd
• KD = kd/ka, KA = ka/kd
o Yes/No Data
• Ligand Fishing
o Concentration Analysis: How MUCH?
• Active Concentration
• Solution Equilibrium
• Inhibition
o Affinity Analysis: How STRONG?
• KD, KA
• Relative Ranking
SPR technology advantages
• Non-label
• Real-time
• Unique, high quality data on molecular interactions
• Simple assay design
• Robust and reproducible
• Walk-away automation
• Small amount of sample required
SPR & ITC combination
ITCSPR
No labeling, no immobilization
In solution
“Elimination” of non-specific
interactions
Thermodynamic and affinity
parameters within one
measurement
Real time measurement
Fast
No labeling, no additional
detection needed
Low sample consumption
Robust
Automatization possible
High sensitivity
Materials for further study
WOULD YOU LIKE TO KNOW MORE?
http://www.sprpages.nl/
Josef Houser
• +420 549 492 527
• josef.houser@ceitec.cz
Michaela Wimmerová
• +420 549 498 166
• michaela.wimmerova@ceitec.cz
bic@ceitec.cz
www.ceitec.cz/z4
Core Facility: Biomolecular
Interaction and Crystallization