Biomolecular interactions - Introduction S2004 Methods for characterization of biomolecular interactions - classical versus modern Bic macromolecules Biomolecules are naturally present in living organisms. Macromolecules. While small molecules consist of up to several hundreds of atoms, macromolecules consist of thousands to millions of atoms. Molecules are basic blocks of matter. They are formed by atoms linked through covalent bonds. Biomacromolecules Biomolecules Saccharides Hormones Pigments Vitamines Alcaloides^ Macromolecules Molecular interactions are central to the molecular basis of life Biomolecular interactions are everywhere... Protein - Ligand Protein - Solvent Protein - Protein Nucleic acid - Solvent Protein - Nucleic acid Nucleic acid - Ligand Protein - Inorganic salt Protein/NA adsorption Nucleic acid - Inorganic salt All processes in living organisms are essentially determined by biomolecular interactions Interaction vs. chemical reaction substrates product □id enzyme enzyme-substrate complex enzyme Interaction vs. chemical reaction ligand 2{I protein protein-ligand complex Antibody - Antigen Receptor - Ligand Transporter - Ligand Lectin - Carbohydrate Transcription factor - Nucleic acid Types of interaction • Nuclear physics interaction of subatomic particles (nuclear phusion, radioactivity) 106 kJ/mol • Chemistry (electron ionization) formation of bonds 150-1000 kJ/mol Biochemistry-biology spectrum of weak interactions (e.g. H-bond 8-30 kJ/mol) Coulombic interactions (salt bridge) Dipole interactions partial charges bond dipoles net dipole Dipole - unequal distribution of electrons in molecule - orientation-dependant Dipole-dipole, dipole-charge, dipole-induced dipole 0» 5h ff 5" J1Ö } Mg2+ 0° ô Nc- R ô+, H 11 Loren Williams o + I V o Loren Williams Hydrogen bonds • Atom with free electron pair + hydrogen bound to electronegative atom (O, N, x, s, c,...) H -H II . N .......o •l M-/ \_M n=< >-n: H o' I G N-H H .-H ^ DNA (base pairing) Antiparallel ß Sheet C'-tcrminus N-termmus - Protein (2D structure stabilization) I II 1 I >f C. ^N-terminus J- -C-tenninus H o- Hydrogen bond Covalent bond HCT OH 0V HO OH—o-' HO. OH HO OH O-f HO" OH O- H / H H H HO OH QJ HO OH / H / H H H HCf OH 0J HO OH O-f H H Polysaccharide (cellulose) Hydrophobic interactions (van der Waals, nonpolar interactions) • Driven by entropy - strong influence of temperature COO" I + H,N—C-H I CH3 Alanine (Ala or A) COO" I +H3N —C—H H3C CH / \ CH, Valine (Val orV) COO" I + H3N-C-H I H—C —CH3 I CH2 I CH3 Isoleucine (lie or I) COO" I +H3N —C —H I CH2 I CH / \ H3C CH3 Leucine (Leu or L) Aromatic stacking (ti-ti interaction) o. 2 I o Phenylalanine Isoleucine Leucine Tryptophan Cysteine Cystine Valine Methionine Tyrosine Alanine Histidine Glycine Threonine Proline Serine Glutamine Asparagine Arginine Aspartic acid Glutamic acid Lysine Mostly more than one effect is present H-bonds Charge/ Acid-Base Hydrophobic/ Steric C10H23 Charge Hydrophobic Interaction description Physical chemistry Why to study the interactions 9 Understanding of biological processes • Does it bind? • How strong the interaction is? • Is the interaction influenced by temperature/additives? Analyzing the nature of intermolecular interaction • What type of interaction is present (hydrophobic, H-bonds, salt bridges)? Application of the knowledge in science/medicine • Disease pattern discovery • Drug development • Biotechnology Receptor - ligand interaction + A NM MX MX r Nx] A A = —Ťi^i Units: L/mol(M-1) , _[MIX] Units: mol/L(M) J^- n — ~~r 1~ is a concentration MX High affinity = large Ka, small Kd Receptor - ligand interaction + MX MX d\MX] _ r^iivi association : —L-^ = ka [M]X] Units: dissociation : = ^ [MX] Units ^ = t.[Mlx]-t„[MX] Receptor - ligand interaction 4mxJ=^[mTx]-^[mx] dt equilibrium: \ d[MX] _ dt = 0 1 _*,=[MIX] MX" KA k Gibbs energy, enthalpy, entropy AG° = -RT In KA = RT\nKD AG° = AH° - TAS° AG < 0 exergonic AH < 0 exothermic AG > 0 endergonic AH > 0 endothermic High affinity = large Ka, small Kd, large -AG0 Enthalpy (H) Entropy (S) Changes in the heat Structure of complex H-bonds Van der Waals Structure of solvent water Changes in the organization Independent rotational and translational degrees of freedom • Complex is more ordered than two free molecules Internal conformational dynamics • flexible molecules lose the entropy upon binding Solvent dynamics • water AG° = AH° - TAS° Rational drug design - Energetic contributions involved > Ligand flexibility Entropy - Hydrophobic interactions - Water release - Ion release - Confromat onal changes Enthalpy - Hydrogen bonds - Protonatioi Hydrogen bonds Hydrophobic parts Water molecules Rational drug design - Energetic contributions involved Why kinetics of interaction is important? KD = K i =*, = [mIx; k [MX -2 r-1 InM = 102s 107 M^s -5 o-l -1 105s 104 M^s -l The same interactions stabilize the protein structure 0-H H .CH2OH N NH V CH. O (a) I C,H; CH. (b) °-H"V°- I H--0 = (CHJ4-NK + (a) CH2COOH O / \ H H Interactions stabilizing the tertiary structure of a (b) protein: (a) ionic bonding, (b) hydrogen bonding, (c) disulfide linkages, and (d) dispersion forces. Ball, Hill, Scott: Introduction to Chemistry: General, Organic, and Biological Methods to study interaction between molecules Optical and spectroscopic methods X-ray, CD, NMR, Mass spektrometry,, fluorescence, IR, Raman... Hydrodynamics sedimentation, ekvillibrium dialysis, capillary electrophoresis, .. Direct measurements AFM, SPR, BLI, piezoelectric biosensors, immunochemistry, microarray... Calorimetry Molecular modelling Experimental techniques to measure the interactions • Physical background • Speed of analysis • Suitable system studied • Availability • Complementarity • "Fashion" Two informational levels of methods Qualitative Ultra violet-visible spectroscopy (UV-Vis) • Absorption spectroscopy in the visible and ultraviolet spectral regions is a powerful technique by which ligand binding equilibria can be studied. u * The peptide bond (absorbs weakly 220 nm) Aromatic amino acids I: Y. W. H i230-300 nm | Many biological molecules show strong absorb uncc in the visible region of the spectrum as a result of the presence of metal ions and prosthetic groups with extended 7T-eleclron systems, such as chlorophyll, carotinoid. flavin, heme. These bands are sensitive to the surrounding polypeptide environment and reflect structural changes, oxidation states, and the binding of ligands. Nienhaus. Karin, and G. Ulrich Nienhaus. "Probing heme protcin-ligand interactions by UWvisihlc absorption spectroscopy." Prote in-Li$tmtl Inter m nous. Humana Press. 200$. 215-241. Fluorescence Resonance Energy Transfer (FRET) Donor and acceptor molecules must be in close proximity (10-100 A). The absorption spectrum of the acceptor must overlap the fluorescence emission spectrum of the donor. • The donor absorption and emission spectra should have a minimal overlap to reduce self-transfer. <10 nm No FRET FRET 405 nm 477 nm 405 nm Hi >10 nm I overlap No FRET Donor Acceptor •rotation I <10 nm FRET Donor Acceptor omiinon •acrteaon From: Broaward ct al. 2013: muure pnaoiols Song. Yang, ct aL "Protein interaction att'inity determination by quantitative FRET technology." BUrteihuttlogy ami bioengineerinx 109.11 4 2012): 2875-2S83. Circular dichroism spectroscopy (CD) • CD is the difference in absorption of left and right circularly polarized light. • Proteins and DNA and many ligands are chiral. • Molecular interactions between chiral and achiral compounds can give rise to induced circular dichroism (ICD) of the achiral counterpart. • It it is chiral then its ICD is the difference between its own CD spectrum and the spectrum in the presence of the protein. 200 220 240 260 Wavelength (nm) Rodger. Alison, ct al. "Circular dichroism spectroscopy lor the stud) of protein-ligand interactions." Pnaein-Ugiuul InwraisiiHts. Humana Press. 2<)()5. 343-363. WlLEYVCH Nuclear magnetic resonance (NMR) spectroscopy • A physical phenomenon in which nuclei in a magnetic field absorb and re emit electromagnetic radiation. • NMR detects ligand binding through changes in the resonant frequencies (chemical shifts) of NMR-active nuclei. • NMR spectroscopy detects and reveals protein ligand interactions with a large range of affinities (10 ^-lO3 M). • Protein samples need to be isotopically enriched (15N and/or 13C). • Larger molecules (>25 kDa), additional enrichment with 2H. • Isotopically labeled protein over-express in bacteria grown in minimal media containing 15NH4C1 and/or 13C glucose as the sole sources of nitrogen and/or carbon. Harald Gunther NMR Spectroscopy Basic Principles, Concepts and Applications in Chemistry Thtrd Edition A • Mittcrmaicr. Anthony, and Erick Mcnescs. "Analyzing Protein--Ligand Interactions by Dynamic NMR Spectroscopy." Pnttein-Ligiuul Interatsioivi. Humana Press. 2013. 243-266. 950 US * Advantage Very sensitive to weak interactions Reveals the portion of molecule involved in interaction Accurate kinetics even for short lifetime bounds (< 1ms) Assay in equilibrium solution Quantitative (large range of affinities i Drawback Needs concentrated isotopically lablled sample 50uM- 2mM) Not suitable for > 100 KDa Needs high purity sample Requires ligand-receptor buffer harmony Strong magnetic fields needed for high quality -> expensive Long assay time Saturation transfer difference (STD) - NMR • powerful method for studying protein-ligand interactions in solution • identifies the binding epitope of a ligand when bound to its receptor protein • Ligand protons that are in close contact with the receptor protein receive a higher degree of saturation in contrast to protons that are either less or not involved in the binding process. • applicable dissociation constants: 10~3-10~8 M • Irradiating the spectral region of broad resonances of the macromolecule which is free of any smaller molecule signals. Electrospray ionisation - Mass spectrometry (ESI-MS) • Using ESI-MS, it is possible to transfer weakly associated complexes from solution into the gas phase inside the mass spectrometer source. • ESI-MS not only provides a direct readout of binding stoichiometry but can also be used to determine dissociation constants ranging from nM to mM. • The number of ligands bound for a given protein-ligand system can be determined directly from the spectrum based on the mass difference between free protein and its ligaled complexes. • In addition to exploiting the \v axis' of the mass spectrum (that is, the mass-locharge ratio, m/z), the *y axis* of the mass spectrum (that is, abundance/intensity) provides important information about affinity and specificity. - I'-nholai /.. Kaitula J.. 1.1 al. Ma%% ^vilfonvtr v t>j>od Wx>l> k> m\olitiak- pio'.an lu'and iiucractKMi.% lof diuir dt>ct*erv.' Chemical Striel\Re\iensA\.\ I <2012): 4335-4355. - HoUifcller.Svwn A.and Kmtm A. Sanitel-Lowery. 'Application* til ESI-MS in diut; diwt*ci> . uitaiot»aU>n olnoncovulcil «.x>niplexei.* Ntiure Renen v Dng Discovery 5.7 < IWb): 585-595. Thermal shift assay (TSA) An increase in the melting temperature of the target protein in the presence of a test ligand is indicative of a promising ligand-protein interaction. High-throughput possibility Fluorescence Po.ik 60000 50000 10000 Dyo Binding Protein Aggregation And Oy» Oissoci.v.on (£ 40000 1 S 30000 jf 20000 Protein Melting I I T 1 T 11 IT T I I I T TT 11 IT M I I T TIM T I T TU IT II I I ! II T Ml ! Ill TU TI I I T TI 11 T 11 I II 25 30 35 40 45 50 55 60 65 70 7 5 8 0 85 90 Temperature (C) £9 SYPRO-ORANGE DYE PROTEIN Temperature Equilibrium dialysis • The molecular weight cut off (MWCO) is chosen such that it will retain the receptor component. • A known concentration and volume of ligand is placed into one of the chambers. The ligand is small enough to pass freely through the membrane. • A known concentration of receptor is then placed in the remaining chamber in an equivalent volume to that placed in the first chamber. • A complete binding curve is generated by measuring Y at different ligand concentrations. • The relationship between binding and ligand concentration is then used to determine the number of binding sites, the ligand affinity, kd. Because this kind of experimental data used to be analyzed with (Scatchard plots) Hatakcyama. TomomiLMi. "Equilibrium Dialysis Using Chromophoric Sugar Derivatives." Let ibis. Springer New York. 2014. 165-171. Affinity capillary electrophoresis (ACE) • The technique uses the resolving power of Cl£ to distinguish between free and bound forms of a receptor as a function of the concentration of free ligand. • ACl£ experiments are most commonly performed in fused silica capillaries by injecting a reeeptor and neutral marker with increasing concentrations ol ligand in the separation buffer. • By studying the mobility change ol a certain molecule when it interacts with another molecule of different mobility it is possible to determine the binding constant between the two compounds. • The binding ol the ligand to the receptor produces a migration time shift in the effective mobility due to a change in the charge:si/e ratio of the complex. • Scutehurd unulysis of the effective mobilities measured as a function of ligand concentration provides the binding aflinil} ol the receptoi ligund complex. - Dingcs. Meredith M.. Kcmal Solakyildirim. and Cynthia K. Larivc. AHinity capillary cIcctrophorcM^ lor the determination of binding affinities lor low molecular weight heparins and antithrombin-III." Elei iroplioresis 35.10 {2014): I 469-1477. - Chen. / la. and Stephen G. Weber. "Determination of binding constants by affinity capillary electrophoresis, clcetrospray ionization mass spectrometry and phase-distribution methods."TrACTrends in Analytical Chemistry 27.9 12008): 738-748. Electrochemical methods • Typically in (bio-)electrochemistry, the reaction under investigation: • Generate current (amperometrie) Generate potential or charge accumulation (Potentiometrie) Alter the conductive properties of a medium (eonduetometrie) between electrodes • Alter impedance • NANO -> The higher surface-to-volume ratio of nano-objects makes their electrical properties increasingly susceptible to external influences. Gricshabcr. Dorothcc. ct al. "Electrochemical biosensors-Sensor principles and architectures." Sen.sors%>3> <2008.i: 1400-1458. Surface plasmon resonance (SPR) • Detection of molecular interaction on a chip surface • Various set-ups: protein-protein, protein-ligand, protein-nucleic acid, protein-lipid membrane, protein-cell/virus Optical Surface plasmon resonance (SPR) Advantage Label free Enables quantitative determination Low sample volume requirement Real time assay Sensitive Drawback Tethering of molecules to surfaces may affect the binding constants measured Any artifactual RI change other than from the interaction can also give signal Stabilization process (in some cases) Cannot verify the stability of the complex formed More tomorrow (Josef Houser) Micro-scale thermophoresis (MST) It measures the motion oi molecules along microscopic temperature gradients and detects changes in their hydration shell, charge or size. An infrared-laser is used to generate precise microscopic temperature gradients within thin glass capillaries that are filled with a sample in a buffer or bioliquid of choice. Thermophoresis, is very sensitive to changes in size, charge, and solvation shell of a molecule and thus suited for bioanalytics. The fluorescence of molecules is used to monitor the motion of molecules along these temperature gradients. The fluorescence can be either intrinsic (e.g. tryptophan) or of an attached dye or fluorescent protein (e.g. GFP). Jcrabck-Willcmscn. Moron, ct al. "Molecular interaction studies using micro scale thcrmonhorcsis." Assay whlilrux tU \ ttt>i»nt/ii leifiHirioKtes9A <2011): 342-353. Initial Temp. Thermo- Steady Back-State Jump phoresis State diffusion ° o ® © _o ©«« © o oOo°© ©°©° o W o ° ©°o° IR-Laser on 20 Time [s] 30 f 40 IR-Laser off 50 cho/cCoid= exP ("St AT) size hydration shell Advantage Drawback Sample concentration (pM/nM) and small volume (< 4 ul) Quantitative (K: pM/nM to mM range and n) measures interactions with essentially no limitation on molecule size or molecular weight. Immobilization free Buffer condition must be absolutely stable Conformational changes induced by IR-Laser heating may be problematic Free in choosing buffer type More later today (Eva Fujdiarova) Isothermal titration calorimetry (ITC) Syringe Reference Cel Ql Sample Cell • All chemical, physical and biologic processes are performed along with heat exchange criteria. • When a protein interacts with a ligand, heat is either released or absorbed. ITC relies only on the detection of a heat effect upon binding -> not relies on the presence of chromophores or fluorophores. Can be used to measure the binding constant, the enthalpy of binding, and the stoichiometrv. Time (min) 30 60 90 3.0 - 6.0 - P + L K, Kŕ Entalpie Afinita Stechiometrie -1-1-1-1-r 0.0 0.5 1.0 PL 1.5 2.0 Molar Ratio AG° = -RTlni: 2.5 A AG° = AH° - TAS° Advantage Drawback Label free Large sample volumes required Enables quantitative determination (K and n) High ligand concentrations Can be done on solutions mat are either homogeneous or heterogenous lYesence of impurities or inactive protein will have a direct impact on the stoic biometry Universal More later today - (Monika Kubíčková) Differential scanning calorimetry (DSC) Measures heat capacity in a range of temperatures. If a ligand binds preferentially to the native state of the protein, the temperature at which the protein-ligand complex denatures will be high compared to the temperature at which the free protein unfolds. Since the degree of stabilization ordestabilization of the native protein depends on the magnitude of the binding energy, comparison of the stability of the complex with the stability of the ligand-free protein allows the binding energy to be estimated. DSC thus provides a direct measure of whether ligand binding to a protein is stabilizing or destabilizing, and so can complement studies of binding equilibria obtained by isothermal titration calorimetry (ITC). Chiii. Michael H.. andElmarJ. Prcnncr. "Differential scanning calorimetry: an invaluable tool for a detailed thermodynamic characterization of macrornolcculcs and their interactions." Journal of Plumtkny AjuI BitmUietl Sciences 3. H 2011): 39. Temperature (° C) Temperature ( °C) Advantage Drawback Label free Sensitivity depends on many parameters Quantitative (relatively) useful in characterizing very tight binding interactions which equilibrate very slowly (mins to hrs) Gives information on the nature of binding event Complex techniques • Indirect detection of molecular interaction • Multi-step approaches Pull-down assay Tandem affinity purification Identification of protein-protein interaction Puig O et al (2001) Methods. Jul;24(3):218-29 TEV recognition Site XZ Tagged protein /~K /~k of interest \J \J \ y*N y—X ) \. Contaminant Associated proteins ) J c )( X ) Elution w/EGTA Associated proteins identified by LC-MS/MS Co-immunoprecipitation MS analysis [1] Addition of antibody to protein extract. [2] Target proteins are immunoprecipitated with the antibody. [3] Coupling of antibody to beads. [4] Isolation of protein complexes. Microarrays • High screening capacity possible • Semi-quantitative 1 MS detection Fluorescence detection V; secondary AB rimary AB POI POI v ,y y yvy ^^ttttttttT ^^tttttttttt/ secondary AB primary AB (A) sandwich tt ttttttttttttt (B) antigen capture (C) RPPA (1 antibody) (D) RPPA (2 antibodies Various immobilized molecules (protein, nucleic acid, saccharide) fixed probes labelled target (sample) L different features (e.g. bind different genes) Fully complementary strands bind strongly Partially complementary strands bind weakly Yeast two-hybrid system Testing for physical interactions between two proteins or protein/DNA. No transcription r.nfjnalP hinrt.ng «it» _ Reporter gene Is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and transcriptional activation. Plasmids encoding two hybrid proteins, one consisting of the GAL4 DNA-binding domain fused to protein X and the other consisting of the GAL4 activation domain fused to protein Y. are constructed and introduced into yeast. Interaction between proteins X and Y leads to the transcriptional activation of a reporter gene containing a binding site for G AL4. Transcription J Cognate binding site Reporter gene • AD: activation Domain • DBD: DNA Binding Domain Reporter gene: LacZ reporter - Blue/White Screening Phage display • For the study of protein-protein, protein-peptide, and protein DNA interactions. • A gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display". • These displaying phages can then be screened against other proteins, peptides or DNA sequences to detect interaction. • The most common bacteriophages used in phage display are M13 and filamentous phage, though T4, T7, and X phage have also been used. Bratkovic. Tomaz. "Progress in phage display: evolution of the technique and its applications." Cellular ami nudemlar life sciences 67.5 < 2010): 749-767. Computational methods • Molecular docking • Virtual screening • Molecular dynamics • Database search > Relatively cheap > Less accurate > Ideally to be combined with experimental approaches Take home message >Many techniques available >Various principles, sample requirements, detection limits.... >There is no single ideal method >Method knowledge is crucial to get the best results Methods for characterization of biomolecular interactions - classical versus modern (autumn 2021) Teachers: Josef Houser, Monika Kubíčková, Jan Komárek, Eva Fujdiarová, Michaela Wimmerová Students enrolled in S2004 course attend only lectures (L). Students enrolled in both S2004 and S2005 courses need to attend both lectures (L) and practical exercises (P). February 3, 2022 (Thursday) time program room time program room 9:00-10:30 L Biomolecular interactions - introduction flt&fRmejrfwá) A5/114 9:00-10:00 P Aiir. _ data analysis (Komárek) A4/217 10:30-10:45 break 10:00-10:15 break 10:45-12:15 L Isothermal titration calorimetry (ITC) ÍKHfeíé&GKá) A5/114 10.15-11.30 P MST - hands-on (Fujdiarová) A4/219 12:15-13:15 lunch 11:30-12:30 lunch 13:15-14:00 L Spectroscopic methods (Houser) 12:30-13:00 P MST - data analysis (Fujdiarová) A4/219 14:00-15:00 L Microscale thermophoresis (MST) (duMOfQXŘ) A5/114 13:00-14:30 P BLI - hands-on (Houser) A4/218 15:00-15:30 break 10:30-10:45 break 15:30-17:00 P ITC - hands-on (Kubíčková) A4/218 15:00-16:00 P BLI - data analysis (Houser) A4/218 16:15-17:00 P Discussion of practical aspects (all teachers) A4/218 time program room 9:00-10:30 L Analytical ultracentrifugation (AUC) (tSm^eJi) A5/114 time program room 10:30-10:45 break 9:30-11:00 Written test A4/211 10:45-12:15 L Surface-based methods (Houser) A5/114 12:15-13:15 lunch 13:15-13:45 L Interactions at the cell level (Houser) A5/114 13:45-14:45 L Importance of sample preparation (Houser) A5/114 14:45-15:00 break 15:00-15:30 P ITC - data analysis (Kubičková) A4/218 15:30-17:30 P AUC - hands-on (Komárek) A4/217