Binding Quantification with

ThermophoresisSeidel S, Dijkman PM, Lea WA, van den Bogaart G, Jerabek-Willemsen

M, Lazic A, Joseph JS, Srinivasan P, Baaske P, Simeonov A, Katritch I, Melo FA,Ladbury JE, Schreiber G, Watts A, Braun D, Duhr S

Speaker: Christian Niederauer

• Introduction• Theoretical Background• Experimental Approach• Signal Analysis

Outline

• microscale thermophoresis • quantifies biomolecular interactions based on

thermophoresis• various molecular properties are influencing MST• low sample consumption• flexible assay design (with/without fluorescent labels)• measurements in cell lysate or complex buffers possible

What characterizes MST?

Theoretical background

Thermophoretic flow: Diffusion flow:

Steady State:

Integration:

Concentration changes due to thermophoresis readout trough measurement of fluorescence

T Tj cD T

Dj D c

0T Dj j

ln( )TS T c hot

cold

e TS T c

c

1d dTD T c

D c

: TT

DS

D

MST Setup

• TC: temperature-controlled tray• OBJ: objective• FO: fluorescence observation• IR: IR-laser• HM: IR-reflecting hot mirror

all-optical approach

Optics Setup

• LED-filter combinations for fluorophore usage :• blue (excitation 460nm-480nm, emission 515-530nm)• green (excitation 515nm-525nm, emission 560-585nm)• red (excitation 605-645nm, emission 680-685nm)

• LED-filter combination for label-free approach:• excitation 280nm, emission 360nm (both UV)

• IR-laser: 1480nm creates temperature gradient volume heated: 2nl by 1K-6K

Fluorescent Labeling

Fluorescent labeling provides high sensitivity:sub-nM concentrations detectable

• fluorescent dye coupled to crosslinker crosslinker binds covalently to functional groups

• non-natural amino acids already carrying a dye

• fusion to a recombinant fluorescent proteins (GFP)

GFP

Intrinsic Fluorescence

• labels may influence binding interactions label-free MST using intrinsic fluorescence

• as low concentration as 100nM possible with > 2 TRP

• quantifiable50nmDK

Tryptophane

Dilution series• non-fluorescent partner titatred against fixed

concentration of fluorescent partner• minimal concentration: unbound state dominant• maximal conc.: saturation of fully bound states ( )20 DK

Capillaries• variation of inner diameter less than 1µm• no diffraction & constant absorption of laser power• constant heat conduction• hydrophilic/hydrophobic coating possible

Signal Analysis

Signal Analysis

I. Initial fluorescence, constant for all samples

Signal Analysis

II. T-Jump due to temperature dependent fluorescence

Signal Analysis

III. Thermophoresis creates concentration gradient

reaches plateau when counterbalanced by mass diffusion

Signal Analysis

IV. Inverse T-Jump

Signal Analysis

V. Backdiffusion compensates concentration gradient

initial fluorescence is nearly recovered

Signal Analysis

1norm

0

ˆ FF F

F Ratio:

Signal Analysis

• . 1norm

0

ˆ FF F

F Ratio:

fraction bound

unbound bound

[ ] [ ]ˆ ˆ ˆ1[ ] [ ]

AB ABF F F

B B

Signal Analysis

• .

unbound bound

[ ] [ ]ˆ ˆ ˆ1[ ] [ ]

AB ABF F F

B B

[ ]ˆ const.[ ]

ABF

B

1norm

0

ˆ FF F

F Ratio:

1)

Signal Analysis

• Law of Mass action:

• with free free

free

[ ] [ ]

[ ]D

A BK

AB free

free

[ ] [ ] [ ]

[ ] [ ] [ ]

A A AB

B B AB

[ ] [ ] [ ] [ ]

[ ]

A AB B AB

AB

2)

Signal Analysis

Solve 2) for fraction bound:

2[ ] [ ] [ ] [ ] 4[ ][ ]

[ ] 2[ ]D DA B K A B K ABAB

B B

Signal Analysis

Solve 2) for fraction bound:

linear

1)

fit

2)

2[ ] [ ] [ ] [ ] 4[ ][ ]

[ ] 2[ ]D DA B K A B K ABAB

B B

[ ]ˆ ~ ~[ ] D

ABF K

B

Plot

• is plotted on linear y-axis in ‰• x-axis is log10 of concentration of titrated partner• sigmoid-shape with bound & unbound plateaus

F̂

Plot

• is plotted on linear y-axis in ‰• x-axis is log10 of concentration of titrated partner• sigmoid-shape with bound & unbound plateaus• is revealed and can be subtracted, getting

• determine by fitting

F̂

unboundF̂

[ ]ˆ[ ]

ABF

B

F̂

Plot

3.8 0.8nMDK