Biomolecular interactions S2004 Methods for characterization of biomolecular interactions - classical versus modern Mgr. Josef Houser, Ph.D. houser@mail.muni.cz Biomacromolecules 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 Macromolecules Composition of biomacromolecules They are formed by linking a huge number of subunits of several types into one chain Macromolecules Building blocks Type of bond Scheme Protein Amino acids Peptidic or 0 ^ ^nh ^nh 0 r Nucleic acid Nucleotides Ester p u\ / o 0H 0// xoh Polysaccharide Monosaccharides Glycosidic oh oh oh Amino acids Acidic Basic Polar Aromatic Hydrophobic H,N Glycine Ö Hj N A til lit iv 'J HjN' Tyrosine O HjN Methionine O H2N Cysteine Ö Aspartic AC id 0 OH H2N Glutamic Acid H2N Vsune O H2N Leucine Ö H2N soleucineÖ H2H" Tryptophan Ö HO,,,/, H2N Threonine Ö H2N Asparagine Ö H2N Glutamine Ö Proline glycine alanine valine leucine isoleucine aspartic acid asparagine glutamic acid glutamine arginine lysine histidine phenylalanine serine threonine tyrosine tryptofan methionine cysteine proline selenocysteine pyrolysine Gly Ala Val Leu lie Asp Asn Glu Gin Arg Lys His Phe Ser Thr Tyr Trp Met Cys Pro Sec Pyr G A V L 1 D N E Q R K H F S T Y W M C P U 0 Biomolecular interactions are everywhere... Protein - Ligand Protein - Protein Protein - Nucleic acid Nucleic acid - Ligand Protein/NA adsorption Protein - Solvent Nucleic acid - Solvent Protein - Inorganic salt Nucleic acid - Inorganic salt All processes in living organisms are essentially determined by biomolecular interactions Interaction vs. chemical reaction substrates product enzyme enzyme-substrate enzyme complex Interaction vs. chemical reaction ligand 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) • Charged atoms = ions • Same charge - repulsion • Opposite charge - attraction Lysine Glutamic Acid Dipole interactions partial charges bond dipoles net dipole • Dipole - unequal distribution of electrons in molecule - orientation-dependant • Dipole-dipole, dipole-charge, dipole-induced dipole Hydrogen bonds • Atom with free electron pair + hydrogen bound to electronegative atom (0, N, x, s, c,...) H Hydrogen bond Covalent bond H „,H- X r)< v N-H---N NT N—H---0 H iO, Ol- DNA (base pairing) OH O OH H ■ O i HO' y- HO OH HO OH .0.--' II -n" .H' Antiparallel ß Sheet 0 H U J. . H OH 0~ . HO OH C-tenninus-' -terminus Protein (2D structure stabilization) N-termiiuis ^ x I II n vC-rermiiius HO OH o OH O hi) Ol H HO OH iO OH .;/ - VÍ : HO Ol- 0 Polysaccharide (cellulose) Hydrophobic interactions (van der Waals, nonpolar interactions) • Driven by entropy - strong influence of temperature COO" I + H3N-C-H I CH, Alanine (Ala or A) COO" I +H3N—C-H I CH / \ H tC CH3 Valine (Val orV) COO" I +H3N-C-H H—C — CH3 CH2 I CH3 Isoleucine (lie or I) COO" I +H3N—C—H I CH2 L H3C CH3 Leucine (Leu or L) Aromatic stacking (k-k interaction) o e 1 Phenylalanine [sofeucine Leucine Tryptophan cysteine cystine valine Metnkonine Tyrosine Alanine Histidine Glycine Threonine Proline Senne Glutamine Asparagine Argmine Aspartic acid Glutamic aci 0 endergonic AH > 0 endothermic 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 loose the entropy upon binding Solvent dynamics • water AG° = AH° - TAS° Why to study the interactions • Understanding of biological processes • Does it bind? • How strong is the interaction? • Is the interaction influenced by temperature/aditives? • 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 Rational drug design -Energetic contributions involved Entropy - Hydrophobic interactions - Water release - Ion release + 4^H^^ - Confromational - water molecules - ions changes Enthalpy - protons - Hydrogen bonds - Protonation The same interactions stabilize the protein structure H .CH2OH N NH. CH. v 9<» I (a) CH. CH_ °-H"\/°-I CH _l_L_ H--0 = {CH2)4—NH3 (a) CH2COOH O / \ H H Interactions stabilizing the tertiary structure of a (b) protein: (a) ionic bonding, (b) hydrogen bhnding, (c) disulfide linkages, and (d) dispersion forces. Ball, Hill, Scott: Introduction to Chemistry: General, Organii, and Biological V Experimental techniques to measure the interactions Classical vs. Modern What is classical? What is modern? Experimental techniques to measure the interactions • Physical background • Speed of analysis • Suitable system studied • Availability • Complementarity • "Fashion" Experimental techniques to measure the interactions p Optical j Fluorescence SPR p--1 MST ATR-FTIR Raman >— NMR uv CD Nnn-npt icu.il Elec trochemistry Equilibrium dialysis J Ch mmatog raphy ITC DSC Electrophoresis MS J PuU-down http://wwwslideshore.net/NoghmehPoonnmohommo/biomoleculor-inte 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. The peptide bond (absorbs weakly 220 nm) Aromatie amino aeids F, Y. Wh H (230-300 nm) Many biological molecules show strong absorb ttiK-c in the visible region of the spectrum as a result of the presCEk.-eof ciietaJ ions and prosthetic groups with extended selection systems, such as chlorophyll, carotinoid, flavin, heme. These hands are sensitive to the surrounding polypeptide environment and reflect structural changes, oxidation states, and the binding ofligands. Nienhaus, Karin, and G. Ulrich Nienhaus. "Probing heme piotein-Jigand interactions by UV/visiblc absorption spectroscopy." Pyo sin-Lsfjti) itJInier man tut. Humana Press, 2ÜÜ3. 2L5-2-41. Fluorescence Resonance Energy Transfer (FRET) * Donor and acceptor molecules must be in close proximity (10-100 A). The absorption spectrum of the acceptor musl overlap the fluorescence emission spectrum of the donor. The donor absorption and emission spectra should have a minima! overlap to reduce self-transfer. DsiiiindKtc No FRET 1 ■1115- nm FREI \ >lDnm Sped ral ovo flap NO FRET Donor Acceptor «bCilriiion m S-1 «10 run FFlET Oonnr Accnplor omnmon ncititwan From: Broitv^jücl ai. 21)L3: tttmttv pYoioculs Siing, Yjnjj, et nl "Prolein interaction affinity deierniination by quantitative FRET technology"Buaedtnoioffy tutti Circular dichroism spectroscopy (CD) ■ CD is the difference in absorption or leť: and right circularly polarized Light. ■ Proteins and DNA and many ligands are chiral. ■ Mo lec u lar i n terac tions betwee n ch iral and ach i rat compounds can give rise to induced circular dichroism (ICD) of the achiral counterpart. * If it is chiral then its ICD is the difference between its own CD spectrum and the spectrum in the presence of the protein. Rodger. Alison, cl al. "Circular dienro ism spectroscopy forthc study o L' protein- ligami interactions." Prtisein-LÍRatití litfertiilit'/ii.-i. Humanj Press, 2M)5. 343-363. WaHtdCunlher Nuclear magnetic resonance (NMR) spectroscopy N M R Spectroscopy Basic Principles,-Concepts and Appti-cation-s in Chemistry Third Edition A physical phenomenon in which nuclei in a magnetic field absorb and re-emil electromagnetic radiation. NMR delects ligand binding through changes in the resonant frequencies (chemical shifts) of NMR-aclive nuclei. NMR spectroscopy detects and reveals protein Ligand interactions with a large range of affinities M). Protein samples need to be isotopically enriched (15N and/or 13C). Larger molecules (>25 kDa)h 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. MiUcimaicr, Anthony, and Ericl Mciksci. "Analyzing Protein-Lijjand Interactions by Dynamic NMR Spectroscopy." Pft/iriH-Lif>nittI Inltrtitjittus. Hununj Pro.t. 21)13. 243-366. Advantage Drawback Very sensitive to weak interactions Needs concentrated isotopically Labi Led sample 50^M- 2mM) Reveals the portion t>f molecule involved in interaction Not suitable for >100 KDa Accurtile kinetics even for short lifetime bounds (< 1ms) Needs high purity sample Assay in equilibrium solution Requires ligand-receptor buffer harmony Quantitative (large range of affinities) Strong magnetic fields needed for high quality -S expensive Long assay lime Mass spectroscopy (MS) * Using ESI-MS, it is possible lo transfer weakly associate J complexes from solution into the gas phase inside the mass spectrometer source. * ESI-M S not on ly prov ide s a direc I re adou I of bi nd ing stoich iometry bu t c an also be used lo 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 Ligated complexes. * In addition to exploiting the \r axis' of the mass spectrum (that is, the mass-tocharge ratio, m/z), the \y axis' of the mass spectrum (that is, abundance/intensity)provides important information about affinity and specificity. Sairiw fUviii WS41.1 I 12012}: 4335^355. I Ivl x.ilki . SV\l-ji \ .. jiiJ Kii.^iii A. S......l--. L-.w..;;. A \lp\n jThmi.% >.l IM MÜ ui dill u Jl-.o^liv: hi;..-: is.. : k-ii ol i ■.... - ^. .iL iL UHi^plexaa." Ntvun Ar views Dnry Dixttnvn $ .1 i ^CKftl: Jftf-iM. 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 ( ■ | ■! i-: a. Surface plasmon resonance (SPR) Advantage Drawback Label free Tethering of molecules to surfaces may affect the binding constants measured Enables quantitative determination Any artifactual RI change other than from the interaction can also give signal Low sample volume requirement Stabilization process (in some cases) Real time assay Sensitive Cannot verify the stability of the complex formed More on Wednesday/Thursday (Josef Houser) Micro-scale thermophoresis (MST) • It measures the motion of molecules along microscopic temperature gradients and delects 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). Jciabck.-'WiHemsen, Moran, ct ul. 'Moleeuljj interaction studies using micnsscule Ihcrmuphurcsis.'" Assa\ atttl tfntg devetoptuenl tecfinafüRiesVA (2U1L): 342-353. Initial Temp. Thermo- Steady Back- "5 State Jump phoresis State diffusion Advantage | Drawback Sample concentration (pM/nM) and Buffer condition must be absolutely small volume (<4ul) stable Quantitative (K: pM/nM to mM range Conformational changes induced by and n) IR-Laser heating may be problematic measures interactions with essentially no limitation on molecule size or - molecular weight. Immobilization free Free in choosing buffer type — Isothermal titration calorimetry (ITC) Syringe Reference Cei Sample Cell ■ All chemicaL physical and biologic processes are performed along with heal exchange criteria. ■ When a protein interacts with a Ligandh heal is either released or absorbed. • ITC relies only on the detection of a heal effect upon binding nol relies on the presence of chromophores or fluorophores. ■ Can be used to measure the binding constant, the enthalpy of binding, and the stoichiometry. Time (min) O 30 60 90 120 p + L „ A » PL Molar Ratio AG° = -RTln KA AG° = AH° - TAS° Advantage Drawback Label free Large sample volumes required Enables quantitative determination (K and n) H igh L i g and conce n Irat ion s Can be done on solutions that are either homogeneous or heterogenous Presence of impurities or inactive protein will have a direct impact on the stoic biometry Universell More on Wednesday- • tomorrow (Eva Dubská) Differential scanning calorimetry (DSC) Measurea heal 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. - - DP Since the degree of stabilization or destabilization 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). Chin. MidiacJ H_ andElmarJ. Premier. "DM'IcrcnlJal tannin jj cahirirnetry: an invaluabJc tool far a detailed thermodynamic chamclerLzalfan of maeramoleculcf and their interactions." Jomnni aj'Pkmrk/ty Ami Scietves 3.1 (2MI): 30. 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 - 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 60000 50000 K 40000 S 30000 jf 20000 10000 Oyv Binding And 0j» Otssiociatior 0 llll|llllll||L|lllllll||L|lllll||||tll1l|IIP|IMMIP|iPIMII|iPIIII| 25 30 35 40 4ä 50 55 «0 65 TO 75 30 85 90 SYPPO-OBANCE DTE Temperature (C> FftOTEIN Temperature Equilibrium dialysis * The molecular weight cui off (MWCO) is chosen such I hat 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 (Scalehard plots) Haiakeyama, Tbmumitsu. "Equilibrium Dialysis Usinjj O*rumoph«ric; Surjar Derivatives." Lectins. Sprimjer New York, 3H4. L65-L7L. Affinity capillary electrophoresis (ACE) 1 The technique uses the resolving power of C\± to distinguish between Tree and bound forms of a receptor as a function of the concentration of free ligand. * ACE experiments are most commonly performed in fused silica capillaries by injecting a receptor and neutraJ marker with increasing concentrations of Ligand in the separation buffer. 1 By studying the mobility change of a certain molecule when it interacts with another molecule of different mobility it is possible to determine the bitiding constant between the two compounds ■ The binding of the ligand to the receptor produces \i migration time shift in the effective mobility due to a change in the charge:size ratio of the complex. ■ Scatehard anaJys is of the e ftecti ve mobi I ities meas tired as a f unetio n of I igand concentratioEi provides the binding affinity of the reeeptor-ligand complex. - Dijicic.-i.. rVtcrcdiih M., Kcmal Solakyildirim, and Cynihia k. Larivc. "Affinity capillary clcetroptandM lor ihe dclcrrnination of binding affinities lor low molecular weight heparins and anlifhrombin-ttt." EUt irt f.v 35.1013)14): 146*?-1477. - Chen, Zhi, and Stephen G. Weber. "Determination of binding constant by affinity capillary electrophoresis, clectrospray ionization mass spectrometry and phase-distribution methods."TrACTrends in Analytical Chemistry 27.9 |20()8): 73K-74H. Electrochemical methods * Typically in (bio^electrochemistry, the re action under investigation: Generate current {amperometric) * Ge nerate pote n t i al or c h arge accu mu 1 at io n {po ten tiome trie) Alter the conductive properties of a medium (conditcrometric) between electrodes Alter impedance * N A NO ^> The higher surface-to-volume ratio of nano-objects makes their electrical properties increasingly susceptible to external influences. Grieshaber, DoiuUkc, ct al. "Electrochemical bicscju,oj3.-5ciwjrprinciples und architectures." SettlorsB3 {2UIHJ): L4^M)- Complex techniques • Indirect detection of molecular interaction • Multi-step approaches Yeast two-hybrid system * Testing for physical interactions between two proteins or prolein/DNA. ■ Is based on the properties of the yeast GAL4 protein, which consists of separable domains responsible for DNA-binding and Iran script ional 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 Yh 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. " AD: activation Domain ■ DBD: DNA Binding Domain * Reporter gene: LacZ reporter - Blue/White Screening Phage display Display variants on surface of phages Analysis Amplify Stock and Titrate Phage Display Cycle 111 Wash Elute * For the study of prole in-proteinh prolein-peptide, and prole in DNA inlerac lions. * A gene encoding a protein of interest is inserted into a phage coal prole in geneh causing the phage to "display". • These displaying phages can then be screened against other proleinsh peptides or DNA sequences lo delect interaction. • The most common bacteriophages used in phage display are M13 and filamentous phage, though T4, T7, and A, phage have also been used. Br.ttoniii. TcniuL "Pn>£rc»> in ph^re dt-pLs. iAuHvm mL lit lÄhinqur ind Lu jpjdK-Jiurfc«.' Criiuht r nr)ti m*Vnirtut tift itttiit n &7.S (3ültl): 749-767. Pull-down assay Tandem affinity purification Identification of protein-protein interaction Puig Oet al (2001) Methods. Jul;24(3):218-29 TEV recognition Sile Tagged protein /-» /^fc of interest \f VJ ^—y Contaminant Associated proteins Associated proteins identified byLC-MS/MS Co-immunoprecipitation [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 H MSdeter.1ion FtuorssDen ce deSeclion (A) sandwich 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 S2004 Methods for characterization of biomolecular interactions - classical versus modern (podzim2014) vyučující: Josef Houser, Eva Dubská, Jan Komárek UTERY 27.1.2015 čas program místnost 10:00-10:15 organizace cvičení A4/211 10:15-12:00 Biomolekulární interakce - úvod (Houser) A4/211 12:00-13:00 oběd 13:00-14:00 Analytická ultracentrifugace (AUC) - úvod (Komárek) A4/211 14:00-15:30 AUC - praktická část - příprava experimentu (Komárek) A4/217 15:30-16:00 přestávka 16:00-16:40 Analytická ultracentrifugace (AUC) - studium interakcí (Komárek) A4/211 16:40-17:00 AUC - praktická část - spuštění experimentu (Komárek) A4/217 - experiment bude probíhat přes noc STŘEDA 28.1.2015 čas program místnost Core Facility: Biomolecular Interaction and Crystallization cular o CO 0 9 Oceitec ^ Josef Houser • +420 549 492 527 • josef.houser@ceitec.cz bic@ceitec.cz www.ceitec.cz/z4 Michaela Wimmerova • +420 549 498 166 • michaela.wimmerova@ceitec.cz