Protein-Protein & Protein-Ligand Interactions (BC4)

Protein-protein & protein-ligand interaction Lars-Oliver Essen MPR-BC Blockseminar/BC4 10-02-2021 First map of protein-protein interactions (PPI) in yeast: >1200 ! Trends in Cell Biology (2001), 11: 102-106 Yeast PPI be...

Protein-protein & protein-ligand interaction Lars-Oliver Essen MPR-BC Blockseminar/BC4 10-02-2021 First map of protein-protein interactions (PPI) in yeast: >1200 ! Trends in Cell Biology (2001), 11: 102-106 Yeast PPI being clustered according to their function Trends in Cell Biology (2001), 11: 102-106 Methods for detecting protein-ligand interactions Curr Opin Biotech (2001), 12:334-339 Qualitative methods: co-immunoprecipation, native MS or PAGE, cross-linking, structural methods, genetics (e.g. Y2H, RNAi, …), ….  Quantitative analysis of binding data Studied system: a priori information about binding partners single-site vs. multiple-site binding expected (e.g. GPCR vs. DNA) stability, solubility, availability of receptors, ligands, proteins Experimental methodology: range of validity, possible artefacts to be countered saturation-binding vs. kinetic method reproducibility of measurements unspecific binding possible Mathematical models: cooperativity possible ? If yes, which types ? non-linear fitting vs. linearized methods error estimates Software: Excel, Prism, Origin, …  Receptor-Ligand Interaction ( P + L PL ) Always known: [P]t, [L]t q = 1. Develop an assay for measuring either [P], [L] or [PL] 2. Plan the experiment with lowest feasible concentration of [P], [L] or [PL]  which is constant ([P]), binding isotherms which is variable ? 3. Perform series of reactions; [L] goes from >KD [L] 4. Equilibrate q = [L] from q = [PL] [P] [L] and KD = [L] + KD [P] + [PL] [PL] 5. Plot [PL] vs. [L]  calculate KD  Issues for calculating KD of ( P + L PL ) If [P]t > KD , then [PL] ~ [P]t , because addition of L only slightly increases [PL]  nothing to be learned, repeat experiment with lower concentrations …  is the method suitable ? Do I have unspecific binding ?  Control experiments, corrected data processing … Is a simple 1:1-binding site model valid ? Check data !  Binding isotherm representation linear  hyperbola semi-log  sigmoidal Hulme & Trevethick BJP (2010), 161:1219-1237  should be symmetric, if ok  pKD is normally distributed, but not KD Do I have enough ligand concentrations; the right ones ? Do I get proper saturation ? Are the data reliable, errors ?  at least triplicates Hill-Diagrams to check for cooperativity P + nL PLn [P] [L]n KD = [PLn] Hill-equation n > 1 cooperative n = 1 non-cooperative log [ q / (1-q)] = n log [L] - log KD n < 1 anti-cooperative  9 Do I have homogeneous binding ? Scatchard plot Like for Lineweaver-Burk plots of Michaelis-Menten kinetics, this plot is usable for quality assessment  data fitting usually done by non-linear fitting Heterogeneity of binding gives non-linear Scatchard plot Example: monoclonal antibodies vs. polyclonal antibodies Non-linear Scatchard plot may also indicate cooperativity other reasons: unspecific non-saturatable binding, experimental errors, …  Methods for quantitative analysis of protein-protein or protein-ligand interaction General: change of some physical quantity upon binding, e.g. heat release/uptake, fluorescence, diffusion coefficient, cross-section, mass, ….  Huge number of methods available, which to choose ? 1. Equilibrium dialysis Dynamic light scattering (DLS) 2. Isothermal titration calorimetry (ITC) EMSA (gel retardation) 3. Surface-plasmon-resonance (SPR) Quantitative ELISA 4. Bio-Layer Interferometry (BLI) RIA 5. Thermophoresis NMR SAXS 6. Fluorescence quench titration 7. Fluorescence anisotropy titration Ki-Determination for inhibitors of competitive catalysis … 1.Measuring Equilibriumaffinity dialysisbyfor equilibrium analyzing dialysis protein-small molecule interaction Simple method, but rather time-consuming Quantitication of bound ligand by isotope-labeling, colorimetric, UV-vis etc. 2. Isothermal-titration calorimetry (ITC) provides thermodynamic binding data - Heat release upon binding is measured by heating up reference cell to same temperature - Only interactions measurable, which have significant enthalpic contribution  ΔH, KD (=ΔG), ΔS ITC Full Tutorial, Microcal. Inc.  Performing the ITC experiment Common problems: high protein conc. needed, heat release due to proton transfer (e.g. to buffer), sensitivity  Characteristic ITC experiment Subtract dilution heats of ligand by using blank titration How data from ITC experiment are evaluated  Time (min) 0 10 20 30 40 5 0 -5 -10 -15 µcal/sec -20 -25 -30 -35 -40 -45 -50 0 -2 kcal/mole of injectant -4 -6 ITC Data Analysis, Microcal. Inc. -8 Data: Rnahhh_NDH Model: OneSites -10 Chi^2/DoF = 2856 N 1.02 ±0.0016 K 5.54E4 ±1.1E3 -12 H -1.361E4 ±29.8 S -22.0 -14 0.0 0.5 1.0 1.5 2.0 Molar Ratio ITC is the only method for resolving the enthalpic and entropic components of binding affinity Caveat: If binding causes proton release/uptake, the heat contribution of the buffer compound has to be eliminated by titrating in different buffer systems. A general problem of all titrations: The right ratio of ligand concentrations tested and KD For ITC: Wiseman value c (= [R]tot / KD ) should be between 1 to 1000 for being able to derive S-shaped binding data (best: 5-20)  Only KD down to 10-8 till 10-9 M measurable, work-around might be displacement ITC or other technique 3. Surface-plasmon resonance (SPR) for analyzing protein-protein interaction Kretschmann configuration Evanescent field ‚senses‘ changes in surface layer SPR sensor surfaces: Linkage chemistries ! Resonance units: proportional to the mass of bound ligand  determine binding at equilibrium with different known ligand concentrations in subphase  KD  Rates of dissociation and association (koff, kon) can be derived ! 4. Bio-Layer Interferometry (BLI) capable to produce similar sets of data as SPR - Sensor tip immersed in liquid records binding by change of thickness at glass-liquid interface - Interference pattern change of white light upon binding can be recorded time-resolved  KD, koff, kon Octet systems: Molecular Devices, USA 5. Microscale thermophoresis (MST): sensitive way for analyzing PPI MST measures fluorescence change upon ligand binding. Two possible causes: a) Migration of fluorescent sample along temperature gradient b) T-related intensity change of fluorescent sample (TRIC)  Very easy to apply, but hard to predict whether given system is suitable for MST Wienken CJ, Baaske P, Rothbauer U, Braun D, Duhr S (2010). "Protein-binding assays in biological liquids using microscale thermophoresis". Nat Commun. 1: 100. Overall procedure of microscale thermophoresis (MST) IR laser heats focal point by 1-10 K Excitation light determines used fluorescent chromophore (Alexa488, flavin, Trp, …)  Theory behind MST, a bit complicated …. … most users don’t understand it anyway Thermophoresis: chot/ccold=exp(-ST ΔT) ≈ 1-ST ΔT ST  Soret cofficient change of sample concentration in focus Recorded signal: ∂/∂T(cF)=c∂F/∂T+F∂c/∂T TRIC effect Given Fnorm = Fhot/Fcold  Linear approx. gives Fnorm ≈ 1+(∂F/∂T-ST)ΔT Processing data by using Fnorm to derive bound fraction q: Fnorm = (1- q) Fnorm(P)+ q Fnorm(PL)  Plot Fnorm vs. log([L]t)  Law of mass action then gives KD  6. Fluorescence quench titration Stern-Volmer Equation (1919) Kq: bimolecular quenching constant [Q]: concentration of quencher t0: fluorescence lifetime if not quenched Jablonski diagram www.chem.vt.edu Meissner et al., JBC 2007  7. Fluorescence anisotropy titration Current Protocols in Chemical Biology 1: 1-15, 2009 Rigider Chromophor, kurze Lebenszeit < 1 ps ||  Mobiler Chromophor, lange Lebenszeit > 1 ns ||  A  Anisotropy; q  Angle between absorbing and emitting dipole; Polarisation 𝑃 = 𝐼|| − 𝐼 𝑰||  Emission parallel to incident light polarization; 𝑰  Emission perpendicular 𝐼|| + 𝐼 Setup for fluorescence anisotropy analysis in 96well plates Assumptions: no quench of reporter fluorophore upon binding Biochemie 4: Zusammenfassung – Methoden PPI/PLI Protein-Protein- und Protein-Liganden-Interaktionen: PPI: Grundlagen & Bedeutung, Dissoziations-/Assoziationskonstante, Übersicht Methoden Bindungsisotherme, 1:1 Bindungsmodell, Scatchard Plot, Gründe für Abweichungen wie Multispezifität & Kooperativität, Hill-Plot Methoden für quantitative Analyse PPI/PLI Gleichgewichtsdialyse Isothermale Titrationskalorimetrie, Grundlagen, Prinzipien der Auswertung & Anwendbarkeit Oberflächen-Plasmonresonanzspektroskopie (SPR, „BiaCore“) Biolayer-Interferometry (BLI), Grundlagen Mikroskalige Thermophorese (MST), Theorie Fluoreszenz-Quenchtitration, anisotrope Fluoreszenztitration, Stern-Volmer-Gleichung Literaturempfehlung: Lottspeich & Engels „Bioanalytik“, 2012, Springer Spektrum 31