S2004 Methods for characterization of biomolecular interactions – classical versus modern MVDr. Eva Fujdiarová, Ph.D. eva.fujdiarova@mail.muni.cz Microscale thermophoresis (MST) Microscale thermophoresis • Method used for determination of the binding affinity of a wide range of interactions • Samples from small ions to big cells • Affinities pM – mM • Little buffer limitation • Small sample consumption • Quick History 1870 Thermophoresis in gas (John Tyndall) Thermophoresis in liquids (Carl Ludwig) First papers on thermophoresis application to affinity of biomacromolecules Thermophoresis in solids (Phillip Schoen) 1856 Thermophoresis in liquids (Charles Soret) 1879 2006 2010 2008 Monolith History 1870 Thermophoresis in gas (John Tyndall) Thermophoresis in liquids (Carl Ludwig) First papers on thermophoresis application to affinity of biomacromolecules Thermophoresis in solids (Phillip Schoen) 1856 Thermophoresis in liquids (Charles Soret) 1879 2006 2010 2008 Monolith Publications dedicated to MST (Pubmed) Thermophoresis movement of particles in temperature gradient Hot Cold ? ? Thermophoresis vs Electrophoresis movement of particles Hot Cold in temperature gradient in electric field + - +-?? Charge, (size)Thermophoretic parameters (Sorret coefficient) A bit of theory… Particle flux j in solution (modified Fick’s law) mass diffusion thermal diffusion ( 𝑗 𝑚) ( 𝑗 𝑇 ) D… diffusion coefficient …particle density DT…thermal diffusion coefficient T…temperature Δ…differenctila value (delta) j = – DΔ – DTΔT Duhr and Braun, PNAS, 2006 At steady state (“equilibrium”), the flux j = 0 thermal diffusion + mass diffusion = 0 Δ =  ⎯ ΔT D DT The difference in molecular density (concentration) depends on: • Used concentration • The temperature gradient • Thermal and mass diffusion coefficitents Duhr and Braun, PNAS, 2006 A bit of theory… At steady state (“equilibrium”), the flux j = 0 thermal diffusion + mass diffusion = 0 ST = ⎯ D DT Thermal diffusion coefficient Mass diffusion coefficient Δ =  ⎯ ΔT D DT Soret coefficient (ST) A bit of theory… Duhr and Braun, PNAS, 2006 At steady state (“equilibrium”), the flux j = 0 thermal diffusion + mass diffusion = 0 Δ =  ⎯ ΔT D DT Soret coefficient (ST) ST = ⎯ D DT A bit of theory… Duhr and Braun, PNAS, 2006 Soret coefficient… for proteins not so easy 𝐴 … surface area of the molecule T… temperature (Kelvins) 𝑠ℎ𝑦𝑑 … hydratation enthropy of the molecule – solution interface 𝜎 𝑒𝑓𝑓 … the effective charge ε … dielectric constant 𝛽 … temperature derivative λ 𝐷𝐻 … Debey-Hueckel length A bit of theory… Soret coefficient ST depends on: • mean temperature • particle size (surface area) • hydration shell entropy (solvation, conformation) • electrostatic potential ( charge) Strength of MST – almost every interaction causes changes in one of these parameters (not in mean temperature) is measurable by MST A bit of theory… MST measurement • Measures fluorescence – one of the binding partners must be fluorescent (e.g. target), constant concentration • Serial dilution of the other partner (e.g. ligand) in capillaries • Two types of lasers • Infrared laser – creates the temperature gradient • ΔT depends on the laser power and time (>10 K after 5 s for 40% laser power) • Excitation laser – excites the fluorescence • Red, blue or green laser • Dye needs to be compatible MST measurement • Capillary scan • Fluorescence for each capillary similar • 10% deviance from average is acceptable MST measurement Fluorescence • MST measurement MST measurement 𝐹 𝑁 = 𝐅 𝐡𝐨𝐭 𝐅 𝐜𝐨𝐥𝐝 Fluorescence Normalized fluorescence FN ,Fnorm • Data analyses MST measurementFluorescence • Data analyses Ligand concentration [µM] Recent recommendationOriginal approach MST measurement • Data analyses Fluorescence Fluorescence Dissecting the MST timetrace Relativefluorescence Time [s] Dissecting the MST timetrace Relativefluorescence Time [s] Heating on (Infrared laser) Heating of (Infrared laser) Dissecting the MST timetraceRelativefluorescence Time [s] Initial fluorescence Bleaching = decrease of fluorescence signal caused by excitation laser • Feature of the dye • Can be induced/influenced by ligand Relativefluorescence Time [s] Dissecting the MST timetrace T-jump • Rapid decrease in fluorescence signal caused by temperature change • Dye dependent • Probes the local surrounding of the dye Relativefluorescence Time [s] Dissecting the MST timetrace Thermophoresis • Molecule flow • Thermal diffusion coefficient DT • Dynamic equilibrium reached at steady state Relativefluorescence Time [s] Dissecting the MST timetrace Mass diffusion • Molecule flow • Diffusion coefficient D Relativefluorescence Time [s] Dissecting the MST timetrace Irreversible effects • Caused by heat demage to the sample • e.g. unfolding, aggregation • Convection Dissecting the MST timetrace Relativefluorescence Time [s] Irreversible effects Mass diffusion Thermal diffusion T-jump Bleaching measures not only thermophoresis but „fluorescence under thermal perturbation“ TRIC – temperature related intensity change NanoTemper, 2018 Dissecting the MST timetrace Relativefluorescence Time [s] Dissecting the TRIC timetrace Sample Size range Sample • Monolith NT measures fluorescence signal • Labelling is necessary unless • You work with fluorescent proteins • GFP (green) • YFP (yellow) • You have Monolith label free instrument • Uses intrinsic flourescence of tryptophanes GFP, PDB: 4OGS Sample Labelling Reactive groups availability: Amino group – Lysine, N-terminus Thiol (sulfhydryl) group – Cystein Sample His-tag Labelling • Dyes compatible with blue, green or red laser • Commercial dyes or specialised dyes from NanoTemper Sample Labelling Which binding partner to label? Interference with interaction 1. Sterical hindrance 2. Conformation changes 3. Non-specific interaction 4. Adhesion to labware 5. Solubility change, aggregation 1. 4. 2. 5. 3. Capillaries • Standard • Coated (premium) • Hydrophobic Which capillary to use? Sample sticking to the capillary wall No sticking A bit of stickingA lot of sticking What can be measured by MST? Affinity KD • What is the strength of interaction? • Labelled partner (target) at constant c  KD • Serial dilution of second partner (ligand) in range of expected KD More than affinity (special cases) • Stoichiometry determination • Multiple binding events within one experiment • Inhibition assay • Thermodynamics measured by MST • Interaction with liposomes • Measurement in crowdy samples (blood, cell lysate) Stochiometry • Labeled partner at constant c > KD • Several dilution of second partner in range of expected molecular ratio Multiple binding events Two independent binding events in one measurement • Labeled partner at constant c  KD,(stronger) • Both KD’s far enough to be distinguishable but close enough to be covered within one dilution row KD = 9.10‒8 KD = 7.10‒6 Inhibition assay • Standard affinity measurement in presence and absence of inhibitor • Comparison of curves / calculated KD Thermodynamics ΔH0 ΔS0/R van’t Hoff plot 0 KD’s • KD determination at various temperatures • Calculation of thermodynamic parameters Enougth theory lets put it into practice Workflow 2. Capillary scan1. Loading capillaries 3. MST measurement 4. Data analyses Relativefluorescence Time [s] Ligand concentration [µM] Fluorescence Capillary position • Optimize sample quality • Sample homogeneity • Pipet more accurately • MST is super sensitive Troubleshooting: fluorescence Standard measurement Direct analysis or Optimization of conditions (additives, buffer, labeling) Troubleshooting: fluorescence SD test • add SDS + DTT mix to first and last sample (lowest and highest concentration) • denature (95°C, 5 min) • check fluorescence Troubleshooting: fluorescence Optimization is necessary: • Centrifuge sample before loading capillary • Add detergents (0.05% TWEEN20, pluronic F-12, BSA) • Optimize buffer composition (pH, salt, additives) Time [s] Relativefluorescence Troubleshooting: aggregation Real hardware MST machines • Monolith NT.115 • Monolith NT.115Pico • Monolith LabelFree • Monolith Automated – all Nanotemper Monolith NT.115 Monolith LabelFree Monolith Automated Monolith NT.115Pico Monolith NT.115 • nM to mM KD range • 16 capillaries (24 in new version) • Two fluorescence channels (BLUE, GREEN, RED) Monolith NT.115Pico • pM to mM KD range • Only RED fluorescence channel Monolith LabelFree • One channel only • Excitation wavelength: 280 nm • Emission wavelength: 360 nm Closest machines: Prague – BIOCEV Vienna – VBCF (Vienna Biocenter) Monolith Automated Two channels possible 96 samples in a run Fragment screening Summary • Thermophoresis is sensitive to subtle changes – almost every interaction will give a signal • Almost every sample that goes in capillary can be measured: ions – cells • Little sample consumption is used (compared to ITC) • Raw data has to be carefully examined for additional effects Summary • Monolith NT measures not only thermophoresis but „fluorescence under thermal perturbation“ TRIC – temperature related intensity change NanoTemper, 2018 • Evaluate MST curves ONLY IF the ligand DO NOT induce: • fluorescence change • bleaching Materials for further study • Ch.J.Wienken et al. (2010), Nature communication Protein-binding assays in biological liquids using microscale thermophoresis • B.López-Méndez et al (2021), Eur. Biophys. J. Microscale Thermophoresis and additional effects measured in NanoTempres Monolith instruments • http://www.nanotempertech.com/ basics, operation manuals, product sheets, explorer community Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz CF Head: Michaela Wimmerová • +420 549 498 166 • michaela.wimmerova@ceitec.cz bic@ceitec.cz bic.ceitec.cz MST at B iomolecular I nteraction and C rystallization Core Facility Eva Fujdiarová • +420 549 497 822 • eva.fujdiarova@ceitec.cz