Surface Plasmon Resonance (SPR) - Biozentrum: Home · In this course we investigate bio-molecular interactions using surface plasmon resonance (SPR). Particularly we would like

Practical exercises 8 Blockkurs Biophysik und Strukturbiologie 2013 L. Kapinos/R. Schoch/R. Lim

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Surface Plasmon Resonance (SPR):

“Probing the structure and activity of proteins by surface plasmon resonance (SPR)”

Biozentrum, University of Basel, Klingelbergstrasse 50-70, 4056 Basel, Switzerland

Dr. Larisa Kapinos

Rafael Schoch

Prof. Roderick Y. Lim

e-mail: [email protected], [email protected], [email protected]

List of Abbreviations 2

1. Introduction 3

2. Materials and method 3

2.1 SPR principle and the Biacore T100 setup 3

2.2 Sensor chips 5

2.3 Preparation of ligand (importin-β or Impβ) and analyte (importin-α or Impα) 5

2.4 Buffer solutions preparation 5

3. Procedures 6

3.1 Sensor chips mounting, docking, functionalizing 6

3.2 Measuring of binding sensograms 7

4. Evaluation of the measured sensograms 9

4.1 Langmuir isotherm (KD) and kinetic analysis (kass and kdiss) for the simple 1:1 binding reaction 8

4.2 Evaluation of the measured sensogram with the Biacore Evaluation software using a

two-state reaction model 9

5. References 9

6. Exercises 10


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List of Abbreviations

Ligand the interaction partner attached to the surface of the sensor chip

Analyte the interaction partner that is passed in solution over the immobilized ligand

BSA bovine serum albumin

HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, buffering agent

EDC 0.4 M 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide

NHS 0.2 M N-hydroxysuccinimide in water

NaCl sodium chloride

RU resonance units

SPR surface plasmon resonance

MW molecular weight


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1. Introduction

In this course we investigate bio-molecular interactions using surface plasmon resonance (SPR). Particularly we would like

to measure the strength of the interaction between importin-α and importin-β. These molecules form a heterodimer in order

to shuttle cargo between the nucleus and the cytoplasm. The binding is expected to be very tight with a binding equilibrium

dissociation constant KD in the low nM range (Catimel et al, JBC, 2001). To validate the binding strength the students are

going to covalently immobilize importin-β to the senor surface using amine-coupling chemistry and subsequently to inject

importin-α at increasing molar concentrations. The resulting binding curve (sensogram) corresponding to the importin-α

binding to the surface anchored Importin-β is then evaluated in order to obtain the equilibrium (KD) and kinetic (kass and

kdiss) binding constants for this complex.

2. Materials and method

2.1 SPR principles and the Biacore T100 instrument setup.

SPR is a common method for the analysis of biomolecular interaction. SPR is sensitive to the changes in the

refractive index occurring at the interface between a thin metal film and a dielectric medium. Binding events are detected

as changes in the solute concentration in proximity to the sensor surface, e.g. for binding to a surface immobilized protein.

Surface plasmon resonance takes place if the wave vector of the incident light parallel to the conductor surface kx matches

the wave vector of the surface plasmon ksp. The wave vector of

the incident light kx depends on the angle of incidence Θ,

whereas the wave vector of the surface plasmon ksp is sensitive

to the refractive index of the dielectric medium in contact with

the sensor surface. Therefore the wave vector of the light kx can

Fig. 1. In SPR systems the incident p-polarized light is wedge-shaped and thus covers a range of incidence angles. The angle at which the maximum loss (sharp “dip”) of the reflected light intensity occurs is called resonance angle or Surface Plasmon Resonance (SPR) angle. The SPR angle shifts when biomolecules bind to the surface and change the refractive index of the surface layer. The SPR angle Θsp is monitored as a change in the position of the intensity dip (from I to II) (taken from Nature Reviews).

Ɵsp


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be tuned by varying the angle of incidence Θ to get ksp = kx. The resonance angle Θsp can be approximated by

where εa and εg are the dielectric constants of the dielectric medium and glass respectively, whereas ε' is the real part of the

dielectric permittivity of the metal. Therefore, this angle varies approximately linear with the refractive index of the

dielectric medium in contact with the senor surface. When molecules in the sample bind to the sensor surface, the

concentration, and therefore the refractive index, at the surface changes and a change in the resonance angle Θsp is

detected. Plotting the resonance angle Θsp versus time during the course of an interaction provides a quantitative measure

of the progress of the interaction. This plot is called a sensorgram.

In the present instrument the resonance angle is detected by the use of a convergent light beam covering a suitable

interval of incidence angles Θ. What the SPR instrument actually measures is the angle of minimum reflected light

intensity that corresponds to the resonance angle Θsp as the energy gets adsorbed by the plasmons. The resonance unit RU

used by the Biacore software represents a change of 0.0001° in the resonance angle Θsp. For most proteins, this is roughly

equivalent to a change in concentration of about 1 pg/mm2 on the sensor surface. The exact conversion factor between RU

and surface concentration depends on properties of the sensor surface and the nature of the molecule responsible for the

concentration change.

The following characteristics of the SPR instrument (Biacore T100, see Fig. 2) should be kept in mind:

- the maximal capacity of the sample loading loop is 350 µl;

- the flow rate of the sample is 1-100 µl/min;

- the temperature range available is 6-40°C (but it should not differ from ambient temperature by more than 10°C);

- four flow cells are available in Biacore T100: one is usually used as a reference cell and the other ones serve for

measuring molecular interactions.

Fig. 2. Setup of the Biacore T100 instrument (GE Healthcare): sensor chips and IFC microfluidic are shown.


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2.2 Sensor chips

As it was mentioned above, the glass surface of the sensor chip is coated with a uniform thin layer of metal (gold,

ca. 50 nm thick) to provide the conditions for surface plasmon resonance. Reliable SPR detection requires a carefully

controlled and uniform metal layer on the glass surface. Usually the gold surface is not a suitable environment to study bio-

molecular interactions. Therefore the sensor surfaces have usually a coating of some sort on the gold layer for specifically

attaching the molecules of interest and protect the biological samples from contact with the gold. For example, GE

Healthcare and some other SPR instruments companies offer a wide selection of the different sensor surfaces to fulfill

varied application requirements for SPR technology including carboxymethylated (CM) dextran matrices, CM-dextran

with immobilized streptavidin, flat carboxylated surfaces (C1 sensor chip) etc.

The SPR measurements during this practical course will be performed using a Biacore T100 automated system

(GE healthcare, Sweden) together with flat-carboxylated surfaces (C1 chip). The sensor chip functionalization is described

below.

2.3 Preparation of ligand (importin-ß or impß) and analyte (importin-α or imp α)

The Ligand (Importin-ß; MW = 97 kDa; OD280 (1%)=7.9) is a molecule attached to the sensor chip. The Analyte

(Importin-α; MW = 59 kDa; OD280 (1%)=7.9) is a molecule, which affinity to the surface-bound ligand is tested. In the

SPR experiments it is important that ligand and analyte solutions are prepared in the exactly same buffer as one used as a

running buffer (buffer used to wash and equilibrate flow cells). It simplifies the sensograms and their analysis, because in

this case any changes occurring in the sensograms would be caused mainly by refractive index changes upon analyte/ligand

molecules injection. Therefore, the proteins (impβ and impα) are dialyzed for ca. 60 min into 500 ml of running buffer (20

mM HEPES, pH 7, 200 mM NaCl, 1 mM MgCl2, 10% glycerol) at room temperature (ca. 25°C). The impα stock solution

after dialysis into running buffer is diluted then to obtain the following concentrations: 10 nM, 25 nM, 50 nM, 250 nM and

500 nM.

A 1 mg/ml solution of bovine serum albumin (BSA) is also prepared using the same HEPES buffer. BSA molecules could

serve as a protection layer in the reference flow cell (no analyte should be able to bind then to the chip surface

functionalized with BSA that forms a inert protein layer in the reference channel of the flow cell (usually channels 1 or 3)).

2.4 Buffer solutions preparation

The buffer (20 mM HEPES, pH 7, 200 mM NaCl, 1 mM MgCl2, 10% glycerol) should be filtered through 0.2 µm

filters and degassed at room temperature.

• All samples >3 mL should be filtered and degassed in the same way.

• All samples <3 mL should be spun at high speed in a mini-centrifuge for 10 min at 25 °C.


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3. Procedures

3.1 Sensor chips mounting, docking, functionalizing

In this project we immobilize our ligand (importin-ß) to the carboxyl groups of the C1 sensor chip using amine coupling

procedure.

Fig. 3. Various coupling procedures to immobilize ligand to the sensor chip surface covalently.

First, EDC/NHS freshly mixed solution is injected into the sample channel to activate carboxyl groups for the amine

coupling of importin-ß (via primary amine or some lysine amines). Then ligand is passed over the surface and the esters

react spontaneously with its primary amine. Afterwards, the reference channel is blocked with BSA molecules also using

‘amine coupling' procedure (see Fig. 3). Finally ethanolamine is flashed though both channels to deactivate excess reactive

groups.

All prepared solutions (0.2 M EDC, 0.4 M NHS, importin-ß and diluted importin-α) should be filled into the special vials

and placed into the Biacore T100 Reagent Rack 1:


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The amount of the immobilized ligand and bound analyte could be estimated using (generally assuming that 1000 RU

corresponds to the binding of 1 ng/mm2)[5]:

3.2 Measuring of binding sensograms

Once the surface has been prepared, the analyte (in our case importin α) can be injected. The following sequence of the

impα concentrations should be used: 10 nM, 25 nM, 50 nM, 250 nM and 500 nM. The recommended flow rate should be at

least 10 µl/min to minimize transport artifacts in the measurements (when binding becomes diffusion controlled).

Eventually, injection of analyte will give no further increase in response when the maximum binding capacity is reached (a

saturation of the binding sites on the chip surface). Such sensogram could then be analyzed.

Req

kass

kdis

t1

t0

Fig. 3. Response in reference (red) and sample (blue) flow cells shown together with the differential sensogram reflecting response change due to the analyte binding to the ligand immobilized on the chip surface.

g nm!"

#$=

1012 ⋅1000 ⋅109 ⋅MW

NA ⋅ΔRU,%where%%

MW%%%,%molecular%weight%of%ancored%molecule,

NA %%%%%,%Avogadro%constant%(6.023⋅1023 %number%of%molecules/mole);

ΔRU%%,%change%of%SPR%signal%upon%binding%of%ligand%or%analyte%


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4. Evaluation of the measured sensograms 4.1 Langmuir isotherm (KD) and kinetic analysis (kass and kdiss) for a simple 1:1 binding (pseudo first-order reaction) For simple 1:1 binding the equilibrium dissociation constant (KD) is defined as:

The association part of the sensogram can be analyzed using the following equation:

where CA is an analyte concentration, kass., kdiss are association and dissociation rate constants, Rt is a binding response at

time t, Req is a binding response at steady-state and Rmax is the maximal binding response. The dissociation part of the

sensogram (washing of the chip surface with running buffer) can be analyzed as follows:

, where R01 is the binding response at the beginning of dissociation.

Req can be plotted versus analyte concentration and this curve can be fitted using a Langmuir isotherm to obtain effective

equilibrium association (KA) or dissociation (KD=1/KA) constants:

Figure 4: Left: Typical differential sensorgram resulting from surface plasmon resonance experiment: Analyte binding with increasing concentrations. Right: Langmuir isotherm fit.

Rt1 = R01 ⋅exp(−kdiss (tn −t1 ))

Req =kass ⋅Rmax ⋅CA

kass ⋅CA +kdiss=Rmax ⋅CA

CA +KD

=Rmax ⋅KA ⋅CA

KA ⋅CA +1

A+Lkass! →!kdiss

← !! AL,!!

at!steady!state!(quasi!equilibrium):

d AL$%

&'

dt= kass A$% &

' L$% &'−kdiss AL$%

&'= 0 ,

KD =A$% &' L$% &'AL$%

&'=kdisskass

Rt =kass ⋅Rmax ⋅CA

kass ⋅CA +kdiss(1−e(−(kass⋅CA+kdiss )(t−t0)) ) = Req(1−e

(−(kass⋅CA+kdiss )(t−t0)) ),


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4.2 Evaluation of the measured sensogram with the Biacore Evaluation software using a two-state reaction model

The binding of importin-α to importin-ß immobilized on the sensor surface was described as a two-state reaction, where the

IBB domain of impα first binds impß and then undergoes conformational change strengthening this complex (Catimel et al,

JBC, 2001) (Fig. 5). Therefore the binding sensogram obtained in our experiments would be also analyzed using a two-

state reaction model imbedded into the Biacore Evaluation software.

Fig. 5. Binding of importin-α to importin-ß immobilized on the sensor surface

5. References

1. Bayliss, R. Littlewood, T., Strawn, L.A., Wente, S. (2002) GLFG and fxFG Nucleoporins bind to overlapping sites of

importin-ß. JBC, 277, 50597-50606

2. Peters, R. (2009) Translocation through the nuclear pore: Kaps pave the way. Bioessays. 31, 466-477.

3. Yamada, J. et al. (2010) A bimodal distribution of two distinct categories of intrinsically disordered structures with

separate functions in FG nucleoporins. Mol. Cell Proteomics (ahead of print).

4. Peleg O., Lim R.Y. (2010) Converging on the function of intrinsically disordered nucleoporins in the nuclear pore

complex. Biol. Chem. , 39, 19-30.

5. Schasfoort, Richard B.M.; Tudos, Anna J. (2008). Handbook of Surface Plasmon Resonance, 401 p.,

6. Schuck P., Minton A. P. (1996) Kinetic analysis of bioensor data: elementary tests for self-consistency. Trends Biochem

Sci., 21, 458-60.

7. J. Tetenbaum-Novatt and M.P. Rout (2011)

8. Catimel et al, (2001) Biophysical characterization of interactions involving importin-alpha during nuclear

import. JBC, 34189-98


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6. Exercises

1. What determines protein-protein interactions? How strong could it be? What is the affinity constant?

2. Estimate how much ligand (importin-ß) was immobilized on the chip surface in your experiment? (molecules/mm2)

3. Determine equilibrium dissociation constant (KD) for importin-α - importin-ß complex at 25°C.

a) What is the Gibbs free energy for this complex at the standard conditions?

b) Determine equilibrium binding constant (KA) for this complex formation at 25°C.

c) Calculate how much of analyte [A] one would need to reach 30, 50 and 99 % binding sites occupancy (Req/Rmax)

at steady state level ?

d) Calculate how much time one needs to reach 99 % of the steady-state level if

[A]=KD and dissociation-rate constant (kdiss ) for this reaction is: 10-1 , 10-2 and 10-3 s-1.

4. Determine kinetic constants for this interaction (kass and kdiss).

5. Determine a stoichiometry of the formed complex if response for the immobilized ligand is 1000 RU and for bound

analyte it is 500 RU. A molecular weight (MW) of analyte is 30 kDa and MW of ligand is 60 kDa.

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Surface Plasmon Resonance (SPR) - Biozentrum: Home · In this course we investigate bio-molecular interactions using surface plasmon resonance (SPR). Particularly we would like