Calorimetry and its applications to Biological Molecules Lise Arleth, Professor BioNano-Science Group, University of Copenhagen, Faculty of Life Sciences Denmark Thanks to Prof. Peter Westh, RUC, for several of the slides in this talk EMBO course in Beijing, April-May, 2011 Dias 1
Calorimetry is (probably) one of the oldest analytical techniques?? Antoine de Lavoisier ’s equipment ~1780 Life processes are a type of combustion EMBO Course, Beijing, April-May, 2011 Dias 2
Measuring principles Detect temperature - calculate heat, Q (= ΔΕ +P Δ V= Δ H) For constant pressure, P: heat=enthalpy change ( Δ H). For constant volume, V: heat=internal energy change ( Δ E). EMBO Course, Beijing, April-May, 2011 Dias 3
Measuring nano-J heats ! All biocalorimeters are “coffe cup” instruments (i.e. measure Δ H rather than Δ E) – (So we allow the sample volume to change slightly) Two (simple) principles: Heat conductor Insulato Insulator r SINK HEAT HEAT SINK Thermo- electric element EMBO Course, Beijing, April-May, 2011 Dias 4
Two types of calorimeters dominate biochemical applications Differential Scanning Calorimetry (DSC) Isothermal Titration Calorimetry (ITC) DSC ITC Measures the heat that is Measures heat of mixing required to linearly increase (titrand into titrate) temperature, T Constant composition – Constant Temperature – temperature perturbed composition perturbed Applications: Applications: Protein denaturation Ligand binding, phase transitions Critical micellar concentrations Protein-surfactant interactions EMBO Course, Beijing, April-May, 2011 Dias 5
Experimental setups: DSC and ITC DSC ITC 200 µ L 500 µ L Sample Sample Shoe-box sized instruments EMBO Course, Beijing, April-May, 2011 Dias 6
Scanning and Isothermal calorimetry DSC: ITC: Measures energy required to Measures energy change of mixing maintain a constant heating (reaction at constant temperature) rate (=C p ) P + L ↔ PL 2’ CMP and 3’CMP binding to RNase Campoy & Freire 2005 State 1 ↔ State 2 EMBO Course, Beijing, April-May, 2011 Dias 7
Bio-calorimetry The pro’s and con’s of application PRO Universally applicable No probe/no special sample preparation Quantitative Non specific CON No structure information Moderate sensitivity Low through-put Non specific EMBO Course, Beijing, April-May, 2011 Dias 8
Differential Scanning Calorimetry EMBO Course, Beijing, April-May, 2011 Dias 9
Assumption: N D K= [D]/[N] T m : Temperature where K=1 ([D]=[N] Δ H: Enthalpy of transition (total area using “step” shaped baseline) Δ S°: At T m : Δ G°=0 hence Δ S°= Δ H/T Δ C p : D-N difference in heat capacity. d Δ H/dT= Δ C p EMBO Course, Beijing, April-May, 2011 Dias 10
Check your assumption: The Van´t Hoff analysis Divide the peak area into T-partitioned slices Determine the equilibrium constant at each temperature E.g. At 50°C: fraction denatured = red area/total area Native fraction (total area-red area)/total area Hence: K(50°C)= red area/(total area – red area) Plot calculated ln(K) values against 1/T. The slope is - Δ H°/R Van't Hoff equation ⎛ ⎞ ⎡ ⎤ ln K 2 ⎟ = − Δ H m 1 − 1 = − Δ H o d ln K ⎜ ⎢ ⎥ K 1 R T 2 T ⇒ ⎣ ⎦ ⎝ ⎠ 1 d (1 T ) R If the Van’t Hoff analysis does not give you this, then your EMBO Course, Beijing, April-May, 2011 assumptions must be wrong (two-state model, baseline or ?) Dias 11
The protein folding problem Molecular interpretations of DSC thermograms • Hydrophobic driving forces • Cooperative units • Quantitative interpretations of mutation-effects • Docking and ”structural thermodynamics” -Has led to an significant part of our current (fragmentary) knowledge on the protein folding process EMBO Course, Beijing, April-May, 2011 Dias 12
Interactions of proteins and other molecules affects the thermogram 2’ CMP binding to RNase Campoy & Freire 2005 The binding of a ligand to the native state brings about stabilization – The dicplacement of the peak along with the change in transition enthalpy quantifies the binding strength EMBO Course, Beijing, April-May, 2011 Dias 13
DSC and lipid phase diagrams EMBO Course, Beijing, April-May, 2011 Dias 14
Alcohols depress the main (P β – L α ) phase transition temperature So does pressure – Le chateliers principle! EMBO Course, Beijing, April-May, 2011 Dias 15
Isothermal Titration Calorimetry EMBO Course, Beijing, April-May, 2011 Dias 16
Isothermal titration calorimetry: Q peak , i = V ⋅ Δ H ⋅ Δ L i = the area under the i 'th peak V : Sample volume Δ H : The characteristic binding enthalpy for the reaction Δ L i : The increase in number of saturated binding sites ⎛ ⎞ Which allows for [ ] i [ ] i − 1 K a L K a L [ ] × Δ L i = P − ⎜ ⎟ determining the [ ] i [ ] i − 1 1 + K a L 1 + K a L ⎝ ⎠ binding constant, K a EMBO Course, Beijing, April-May, 2011 Dias 17
Limitations of measurements Window of binding strength typically 10 3 -10 9 M -1 Use Competition-binding assays to get up to 10 12 M -1 Too strong Perfect Difficult Too weak Advantages of ITC measurements High resolution Fast Several binding parameters in one trial EMBO Course, Beijing, April-May, 2011 Dias 18
Elaborated ITC Example: Protein-surfactant interactions Collaboration with P. Westh, L. Lundby-Hansen Surfactants (=detergents) Proteins • Amphiphilic • Hierachical structure • Selforganize into micelles • Folded / Unfolded when surfactant concention exeeds critical Monomer Concentration (cmc) = ? + Practical relevance: Detergent Enzyme industry EMBO Course, Beijing, April-May, 2011 Dias 19
Surfactants and the critical micellar concentration (CMC) Surface tension, γ (mJ/m 2 ) Air Water Air Water Critical micellar concentration Ln (Concentration) EMBO Course, Beijing, April-May, 2011 Dias 20
ITC – Typical data set: -As obtained in Prof. Peter Westh’s Lab., Denmark Raw data ITC data HiC protein (an enzyme) titrated with the detergent SDS EMBO Course, Beijing, April-May, 2011 Dias 21
Demicellization versus temperature Δ H demic is T-dependent => We can “contrast- match” it out at a given temperature Critical micellar concentration Δ H demic = 0 at 22ºC CMC SDS = 2.2 mM at 22ºC Buffer: 50 mM TRIS, 2 mM EDTA, pH=7 EMBO Course, Beijing, April-May, 2011 Dias 22
ITC-scans of protein-surfactant interactions at 22 C SDS-HiC ( Humicola insolens SDS-BSA ( Bovine Serum Albumin ) pisi cutinase ) Data suggest that there is more information than Tanfords ”Each g protein binds 1.4 g SDS”: ”Thermodynamic fingerprint” EMBO Course, Beijing, April-May, 2011 Dias 23
Complementarity between SANS/SAXS and ITC SANS ITC Very detailed structural Measures of enthalpy of information can be surfactant-protein obtained interactions. ”Thermodynamic fingerprint” Time consuming, requires large facility, 1 sample takes 2 hours at the Small laboratory based SANS-II at PSI. Data instrument, 1 full titration analysis may be relatively scan takes about 3-6 hours complicated No structural information EMBO Course, Beijing, April-May, 2011 Dias 24
Focus: SDS:BSA system and its thermodynamic finger print. At the very beginning ….. C : ? A B C D E D : ? E : Saturation A : Specific binding B : ? EMBO Course, Beijing, April-May, 2011 Dias 25
Vary BSA concentration SDS concentration Plot as a function of SDS-BSA Molar ratio EMBO Course, Beijing, April-May, 2011 Dias 26
Concentration dependence of titration scans Identify characteristic points EMBO Course, Beijing, April-May, 2011 Dias 27
Intercept at [BSA]=0 gives free monomer concentration at given point, Slope gives binding number EMBO Course, Beijing, April-May, 2011 Dias 28
Binding Isotherm determined from ITC Realistic ? EMBO Course, Beijing, April-May, 2011 Dias 29
Performed SANS measurements along the titration scan C : ? A B C D E D : ? E : Saturation A : Specific binding B : ? EMBO Course, Beijing, April-May, 2011 Dias 30
SANS data (pure sds, pure bsa and mixtures) I(q) Step 1: Use this for determining the forward Scattering I(0), (via IFT) p(r): Pair Distance Distribution function EMBO Course, Beijing, April-May, 2011 Dias 31 SDS BSA BSA:SDS MR=50
Binding Isotherm determined from ITC and from SANS So our ITC point-plot-method gives the same binding isotherms as we obtain from SANS EMBO Course, Beijing, April-May, 2011 Dias 32
Thermodynamic finger print and structure of surfactant protein complexes C : 1st unfolding, Size of complex and A C free increases B E C D D : 2nd unfolding Further elongation of complex, C free increases weakly E : Saturated complexes, monomers and micelles A : Strong Specific binding Nbinding at saturation: 210 SDS per BSA, =0.9 g SDS per g BSA B : Strong increase of C free – Weak increase EMBO Course, Beijing, April-May, 2011 of binding number Dias 33
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