S2004 Methods for characterization of biomolecular interactions – classical versus modern Mgr. Josef Houser, Ph.D. houser@mail.muni.cz Spectroscopic and related methods Spectrum of light • UV/Vis spectroscopy • Fluorescence spectroscopy + Fluorescence resonance energy transfer (FRET) • Circular dichroism (CD) • Static and Dynamic light scattering (SLS, DLS) • Fourier transformed infrared spectroscopy (FTIR) + ATR-FTIR – attenuated total reflectance • Surface enhanced Raman spectroscopy (SERS) • Nuclear magnetic resonance (NMR) • Surface plasmon resonance (SPR) • Micro-scale thermophoresis (MST) Spectroscopic methods UV/VIS absorption UV/VIS spectroscopy Wavelegths: UV 180 – 350 nm (160 – 380 nm) VIS 380 – 750 nm Absorption spectroscopy – absorption of light of given wavelength Proteins – 250-300 nm – aromatic AA <220 – peptide bond >300 nm – colourful proteins Nucleic acids – 240-300 nm UV/VIS spectroscopy Coloured proteins: • Special amino acid arrangement – e.g. GFP • Prosthetic groups – heme, flavin,... Pakhomov 2008, Cell Heme FMN UV/VIS spectroscopy Analysis of absorption spectra with and without ligand Binding in Trp proximity distinguishable Mainly for relatively low concentrations (nonlinearity at high OD) Interaction of protein at high density (Ab drugs) – based on non-ideality in spectra measured UV/VIS spectroscopy Hemoglobin – interaction with O2, drugs/inhibitors Spectrum changes higher for chromophore proximity Xu2019,SpectrochimicaActaA chegg.com Hemoglobin – levamlodipine UV/VIS spectra measurement Spectrometers • Various wavelength range • Single / Dual beam Cuvettes • Optical glass (VIS) or Quartz (UV) • Fixed path length (0.01 – 10 mm) • Demountable cuvettes – lower accuracy Fluorescence Fluorescence spectroscopy Fluorescence • Excitation – higher energy  shorter wavelength • Emission – lower energy  longer wavelength (red shift) Fluorescence spectroscopy Fluorophores • Dyes • Fluorescent proteins (GFP, YFP,…) • Tryptophan – intrinsic fluorescence (ex)  280 nm (em)  350 nm FITC (fluorescein isothiocyanate) (ex)  495 nm (em)  519 nm Tryptofan Fluorescence spectroscopy Influence on fluorescence • Environment polarity • Solvent viscosity • Probe conformational changes • pH (protonation) • … (ex)  280 nm (em)  350 nm Trp – free Trp – BSA Trp – OVA Moller 2006 Fluorescence spectroscopy • Change of fluorescence upon ligand binding 0 0.1 0.2 0.3 0.4 0.5 290 340 390 440Intensity[A.U.] Wavelength [nm] 0 0.1 0.2 0.3 0.4 0.5 290 340 390 440 Intensity[A.U.] Wavelength [nm] 0 0.1 0.2 0.3 0.4 0.5 290 340 390 440 Intensity[A.U.] Wavelength [nm] 0 0.1 0.2 0.3 0.4 0.5 290 340 390 440 Intensity[A.U.] Wavelength [nm] Fuc αMeFuc L-Gal ManSite 2 Lectin AFL Fluorescence spectroscopy 0.400 0.410 0.420 0.430 0.440 0.450 0.460 0.470 0.480 0.490 0.500 1.E-6 1.E-5 1.E-4 1.E-3 1.E-2 1.E-1 Fluorescence[a.u.] log cligand MeFuc L-Fuc L-Gal D-Man 331 332 333 334 335 336 337 338 339 340 341 1.E-6 1.E-5 1.E-4 1.E-3 1.E-2 1.E-1 Em.max[nm] log cligand MeFuc L-Fuc L-Gal D-Man 0.000 0.001 0.002 0.003 0.004 0.005 0 2 4 6 8 Data: AFL1 Fucose Model: OneSiteBind2n Equation: y = Bmax * x / (k1 + x) Weighting: y No weighting Chi^2/DoF = 0.11193 R^2 = 0.98305 Bmax 6.88138 ±0.22946 k1 0.00013 ±0.00001 c(Fuc) [M]  Total fluorescence (quantum yield)(max) Properties to analyze: • Excitation/emission maximum • Quantum yield – fluorescence intensity Fluorescence anisotropy = fluorescence polarization • Change of fluorescence polarization upon interaction • Faster for smaller particles • Influenced by viscosity and temperature horiba.com BMG Labtech Fluorescence anisotropy • Two ways of analysis: • Steady state – various ligand conc. • Anisotropy decay over time • Fit of data by binding curve • Free vs. bound ligand – FA difference horiba.compicoquant.com FRET (Fluorescence resonance energy transfer) Two fluorescent dyes • Dye 1 excited by specific wavelength • Dye 1 emission is able to excite Dye 2, when close • Dye 2 emission is measured Pehlivan 2019, Microchimica Acta FRET (Fluorescence resonance energy transfer) Sasaki & Yoshida 2016, Drug Discovery Today: Technologies Example: Tracking of bromodomain/ histone interactions in cells Example: FRET-based detection of SUMO1 and its E2 ligase, Ubc9, interaction. Song 2010, Annals of Biomedical Engineering Fluorescence measurement Fluorimeters – dedicated spectrometers Monochromator/filters Excitation and emission spectra 90° fluorescence measured Cuvettes Quartz or optical glass Coupled to imaging – fluorescence microscopy Labeling intrinsic, chemical in situ, co-expression portal.faf.cuni.cz Circular dichroism Circular dichroism spectroscopy (CD) • CD is the difference in absorption of left and right circularly polarized light • Proteins and nucleic acids are chiral = CD active chem.libretexts.org ΔA = AL − AR = Δεcl = (εL−εR)cl CD spectroscopy • Frequently used to determine 2D structure • Specific absorption curves • Induced circular dichroism – caused by interaction between chiral and achiral compound • Differential CD spectra analyzed CD spectroscopy • Full/Partial folding/unfolding of protein upon interaction – IDPs • Sometimes even minor changes can be detected Example: Molybdate-sensing protein ModE in absence (solid) and presence (dotted) of molybdate S.M. Kelly et al. / Biochimica et Biophysica Acta 1751 (2005) 119 – 139 CD measurement • Dedicated CD spectrometers • Mostly UV region • Cuvettes from quartz • Buffer absorption • Sample purity importance J-815 (Jasco) Light scattering Light scattering • Light scattering depends on size of particles in solution • Static light scattering • Intensity of scattered light • Proportional to molecular mass • Dynamic light scattering • Time fluctuations of scattered light • Proportional to molecular size Static light scattering (SLS) • Multi-angle light scattering (MALS) • Analysis of set of samples with various composition – concentration gradient (CG-MALS) • Separation of individual species coupled to LS – SEC-MALS, FFF-MALS Theoretical SLS signal for different protein Y – protein X ratios. Some 2013, Biophys Rev 2:1 3:12:2 1:1 No interaction Dynamic light scattering (DLS) • Low resolution • Protein-protein or protein-NA interactions 1:1 α-chymotrypsin/bovine pancreatic trypsin inhibitor interaction. The profile expected for no association is shown by the dotted line. (Inset) Negative control of α-chymotrypsin and lysozyme. Infrared and Raman spectroscopy FTIR (Fourier transformed infrared spectroscopy) All biomolecules absorb in infra red region – vibration of chemical bonds: Wavelength  = app. 1 – 50 μm Wavenumber ෤𝜈 = 10 000 cm–1 – 200 cm–1 Strong absorption by water Analysis of IR spectra of free components and the complex ATR-FTIR (Attenuated total reflectance FTIR) • Molecule of interest is present near the sensor surface • Signal of water is highly reduced • Higher sensitivity, lower concentration needed • Surface bound receptor – ligand is detected only upon binding SERS (Surface enhanced Raman spectroscopy) • Raman spectroscopy – analysis of vibrational/ rotational/etc. states in system through scattered light • Very weak • Strong enhancement (102 – 1014) by adsorption on surface of metal or semiconductor Raman vs. Infrared spectroscopy Light as a tool Optical tweezers • Manipulation of molecules/microscopic objects by light • Focused laser beam • Combined with microscope • Objects up to micrometer size – living cells Tweez250 (Quantum Design) Bustamante 2021, Nature Optical tweezers – applications Systems: • Protein-protein • Protein-NA • Nanoparticles • Cells Features: • Protein folding • DNA stability • Interactions • Binding forces Bustamante 2021, Nature Spectroscopic and related methods • Various use of light – absorbance, fluorescence, scattering • Broad range of wavelengths • Intrinsic properties vs. specific labeling • Level of description of interaction • Detection • Quantification – KD, KA • Detailed description – interaction forces 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 Biomolecular I nteraction and Crystallization Core Facility