BIOL 25012 2022-23 Advanced Biochemistry - Ligand Binding

Module BIOL 25012 2022 - 23 Advanced Biochemistry WEEK 32 Ligand binding and binding energy: BIOCALORIMETRY ALDO 26 April 2023 1 Ligand binding and binding energy: BIOCALORIMETRY LEARNING OUTCOMES. At the end of this lecture you should be able to: • Describe the technical aspects...

Module BIOL 25012 2022 - 23 Advanced Biochemistry WEEK 32 Ligand binding and binding energy: BIOCALORIMETRY ALDO 26 April 2023 1 Ligand binding and binding energy: BIOCALORIMETRY LEARNING OUTCOMES. At the end of this lecture you should be able to: • Describe the technical aspects behind Isothermal Titration Calorimetry (ITC) • Explain how a complete thermodynamic characterization of ligand binding can be obtained from a single ITC experiment: D H B , K eq , N , D G B , D S B • Evaluate and discuss advantages and disadvantages of ITC technique. A B FUNCTION A common pattern in biology : Recognition ➔ Binding ➔ Function (slides from last week’s BIOL22081 lecture) Measuring ligand binding energy A reminder from BIOL14407 Molecular principles for Biochemistry Equilibrium constant ( K c) and free energy ( D G) ∆ ?????? ∅ = − ???????????? ln ?????? ?????? ∆ ?????? ∅ = − ???????????? ln ?????? ?????? R = 8.31 J / mol. K. (universal gas constant) Due to the exponential dependence of K con D G, small changes in D G cause great changes in the equilibrium constant K c From thermodynamics to kinetics: Equilibrium and rate constants Thermodynamics Kinetics In a reversible reaction, chemical equilibrium is reached when the rates of the forward and reverse reactions are equal and the concentrations of the reactants and products no longer change. Equilibrium and rate constants association Therefore, + + + In a binding interaction, the free energy ( D G) is a function of the equilibrium binding constant ( K eq ) Measuring energy from binding in biological interactions: Isothermal titration calorimetry (ITC) https://www.youtube.com/watch?v=o_IpWcWKNXI Calculating binding affinity ( K D ) by ITC Binding is too tight to be measured ligand free ligand saturated D H B K B N D G B = D H B – T D S B K eq = e -D G B/RT All thermodynamic parameters can be determined by ITC: D H B , K eq , N , D G B , D S B . PROTEIN Reference cell LIGAND DT Isothermal titration calorimetry (ITC)-2 -1 0 0 25 50 75 100 Time (min) µcal/sec 0.0 0.5 1.0 1.5 2.0 2.5 -16 -12 -8 -4 0 Molar Ratio kcal/mole of injectant 26 April 2023 17 Self -study material: Running an ITC experiment and its data analysis This animation will NOT be evaluated in the second phase test. However, this material is useful to improve your understanding of ITC 26 April 2023 19 The energetics of binding: enthalpy ( D H) and entropy ( D S) 26 April 2023 20 26 April 2023 21 CASE STUDY: Energetics of NADPH binding to Cytochrome P450 reductase (CPR) pdf file in NOW NADP + ?????? ???????????? = 1 ?????? ?????? K d : dissociation constant NADPH -2 -1 0 0 25 50 75 100 Time (min) µcal/sec 0.0 0.5 1.0 1.5 2.0 2.5 -16 -12 -8 -4 0 Molar Ratio kcal/mole of injectant NADPH 17560 Chi2 -15 -1.2 ±0.1E4 7.3 ±0.5 E5 1.1 ± 0.01 DS DH K N 9978 Chi2 -18 -1.4 ±0.005 E4 1.5 ±0.06 E6 1.0 ± 0.006 DS DH K N 2’5’ -ADP How much does the nicotinamide moiety contribute to binding? Nicotinamide moiety Adenosine moiety 2’5’ -ADP 3’5’ -ADP How specific is substrate recognition? No binding even at 800 molar ratio !!! http://jkweb.berkeley.edu/external/pdb/2005/sondermann_pnas_2005/ Probing binding mechanisms More sophisticated ITC applications Mutant D140A Mutant R153A Detecting multiple binding sites More sophisticated ITC applications Applying ITC for the developing of high affinity anti -HIV drugs Seminar by Prof. Ernesto Freire John Hopkins University (USA) This specific animation will NOT be evaluated in the second phase test. However, this material is useful to improve your understanding of ITC •Complete basic thermodynamic characterization (stoichiometry, association constant, and binding enthalpy) in a single experiment. •Heat is a universal signal, and, hence, no need for reporter labels (e.g. chromophores, fluorophores). •Direct determination of the binding enthalpy. •Non -destructive technique. •Interaction in solution. •Possibility of performing experiment with optically dense solutions or unusual systems (e.g. dispersions, intact organelles or cells). •Considerably fast (from 0.25 h/assay in the single injection experiment to 2 h/assay in the conventional titration experiment). ITC advantages There are however, certain disadvantages: •Signal is proportional to the binding enthalpy, and the non -covalent complexes may exhibit rather small binding enthalpies •Heat is a universal signal and each process contributes to the global measured heat, thus complicating the evaluation of the contribution because of binding •Large amount of sample is needed, though this has come down considerably •It is a slow technique with a low throughput (0.25 - 2 h/assay), not suitable for high -throughout screening. •Kinetically slow processes may be overlooked •A limited range for consistently measured binding affinities (association constants nanoM to microM for the traditional titration experiment) ITC disadvantages Ligand binding and binding energy: BIOCALORIMETRY CONCLUSIONS • Isothermal Titration Calorimetry (ITC) allows direct measurement of the heat evolved ( D H B) during biological interactions. This is achieved through a two -cell system which constantly compare temperatures (i.e. isothermal). • In most biological interactions the bonds formed are non -covalent (i.e. ionic and hydrogen bonds). These interactions are relatively weak and reversible: D G B = 10 – 20 kilocal /mol. • Direct determination of D H B and K eq allows calculation of the entire thermodynamic parameter set: D G B , D SB • ITC’s greatest advantage is the absence of any modification/ labelling neither ligand or receptors. Heat is an universal signature of molecular interactions.