binding quantification with thermophoresis seidel s, dijkman pm, lea wa, van den bogaart g,...
TRANSCRIPT
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