09/11/2010 CALORIMETRIA strumenti ed applicazioni Università di Parma, 9 novembre 2010 R. Pepi TA Instruments Thermal Analysis Thermal analysis refers to a group of techniques in which a physical property of a substance is measured as a function of temperature whilst the substance is subjected to an imposed temperature program under controlled atmosphere Examples: DSC, TGA, TMA, 1
09/11/2010 Calorimetry Calorimetry refers to those measuring techniques that are used for direct determination of rate of heat production, heat and heat capacity as function of temperature and time Examples: DSC, TAM, ITC TA vs. Calorimetry Thermal Analysis Calorimetry TGA TMA DMA DSC TAM ITC Solution Calorimetry Reaction Calorimetry Scanning, Temperature-Induced Processes Scanning or Isothermal 2
09/11/2010 Calorimetry Features All kinds of processes result in either heat production or heat absorption: Chemical, Physical and Biological Non-specific Direct and continous Reaction rate Heat production rate dc = k dt dc P = H dt P = H k f ( c) f ( c) Enthalpy Thermodynamic Information Reaction rate Kinetic Information Concentration Analytical Information 3
09/11/2010 Heatflow vs. Time 18000 Heat flow (µw/g) 16000 14000 12000 10000 8000 6000 4000 2000 0 f(t, P, Q) 0 1 2 3 4 Time (d) Shows how the reaction rate varies with time. P = H dc/dt Energy vs.time Energy (J/g) 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 0 1 2 3 4 Shows how the extent of reaction varies with time. P dt = H dc Time (d) 4
09/11/2010 Heatflow vs. Energy Heat flow (µw/g) 18000 16000 14000 12000 10000 8000 6000 4000 2000 0 0 5000 10000 15000 20000 Energy (J/g) Shows how the reaction rate varies with the extent of reaction (ex. 1 order Q proportional to P with rate const., k, as proportionality constant) Calorimetry Instruments TAM III Isothermal Calorimeter (±0.00005 C) Versatile, Modular System Stability/Compatibility Perfusion Isothermal Titration Calorimetry Solution Calorimetry Nano-ITC Dedicated Isothermal Titration Calorimeter (Protein) Binding (Protein) Interactions Nano-DSC Dedicated Scanning Calorimeter (Protein) Structure/Stability MC-DSC 5
09/11/2010 Calorimetric Range 1 nw -11-9 -7-5 1 mw -3-1 1 3-12 -10-8 -6-4 -2 1 pw 1 µw 0 1 W 2 4 General Features of TAM Thermostat Controls the temperature Calorimeters Contains the heat detectors Sample handling systems Contains the sample Auxiliary equipment Equipment used to control the experimental conditions such as relative humidity, oxygen concentration. Software 6
09/11/2010 TAM: an Integrated System Monitor Calorimeters Thermostat Heat exchange by Peltier coolers Oil expansion tank Keyboard Computer Power Supply Circulation Pump Twin System (used in TAM) Sample cell (S) Surrounding (heat sink) T o Surrounding (heat sink) Reference cell (R) k S k R Sample holder P s T S C S Φ S Sample holder Pr TR C R Φ R T o T=(T s -T o )-(T R -T o )=T s -T R 7
09/11/2010 Heat Balance Equation P Φ To General Heat Balance Equation P = Φ + dt C dt TS Rate of Heat Production = Heat flow + Rate of Heat Accumulation or depletion TS dt C dt High Sample Throughput TAM is a multichannel microcalorimetric system offering up to 48 experiments to be performed. TAM is ideal for research purposes as well as for large scale screening 8
09/11/2010 TAM Calorimeter Features All kinds of processes; Chemical, Physical and Biological Non-specific Non-destructive (sometime) Not dependent on the physical shape of the sample No need for sample preparation Direct and continous 4-ml Nanocalorimeter Sample Ampoule holder Twin System Thermoelectric Modules (Seebeck modules) Foil Heaters for Calibration Reference Ampoule holder Heat Sink Partition wall 9
09/11/2010 Minicalorimeter (4 ml) Sample Ampoule holder Outer Steel Cylinder Thermopiles Heat Sink Reference Ampoule holder Sample Handling Systems Closed or sealed (static) Ampoules Open ampoules - Micro Reaction System Micro Solution Ampoule 10
09/11/2010 TAM Stability Testing Heat Flow exo Least stable Most stable X X X Time Drug Efficacy Flow calorimetry: Leukemia (T-lymphoma) cells exposed to the anticancer drug methotrexate. The final drug concentrations were (a) 0, (b) 0.2, (c) 0.5, (d) 1.0, (e) 2.0, (f) 4.0µM (ref 6). a b c d e f Bermudez, Backman and Schon., Cell. Biophys. 20, 111-123, (1992). 11
09/11/2010 Response of Streptococcus Mutants Flow calorimetry: in Absence and in Presence of an Antimicrobial Agent Morgan, Beezer, Mitchell and Bunch, J. Appl. Microbiol. 90, 53-58, (2001). Oxidation of Meclofenoxate Hydrochloride 50 C Heat Flow (µw) 40 C 30 C Tomoko Otsuka, Sumie Yoshioka, Yukio Aso and Tadao Terao, National Institute of Hygienic Sciences, Tokyo, Japan, Chem. Pharm. Bull. 42(1) 1994 23 C 12
09/11/2010 Nano-ITC Specifically designed to measure molecular interactions and reaction kinetics. Capable of thermal measurements over a wide variety of solution conditions and temperatures. In-solution universal detector for maximum data collection in a single experiment. ITC Applications Molecular Binding Studies Quick and accurate affinities Structure-function relationships Affinity and mechanism of action screening Specific vs. non-specific binding Quality and process control Protein engineering assessment Validate virtual models Drug resistance and adaptation evaluation Reaction Kinetics K M, V max, k cat Enzyme Inhibition 13
09/11/2010 Nano ITC Schematic A. Heating and cooling TED B. Thermistor C. Cylindrical cells D. Constant cooling E. Power compensation heaters F. Thermal shield G. Thermosensor H. Syringe stepper motor I. Temperature control block J. Temperature measurement K. Power compensation L. Signal amplifier M. Feedback control algorithm N. Temperature control algorithm O. Syringe control algorithm Nano ITC Cell Design Conical tops and bottoms promote consistent bubble free filling, uniform stirring and easy cleaning. Cylindrical cells are made of chemically inert 99.999% pure gold. 14
09/11/2010 Nano ITC Syringe & Stirrer Design Embedded linear actuator Threaded syringe mount Twisted stirrer paddle Spring loaded electronic connections for wire free operation Isothermal Titration Calorimetry 7 Typical ITC Data 6 5 Heat Rate / µw 4 3 2 1 0-1 0 1000 2000 3000 4000 5000 6000 Time / s ITC Strength: Direct measure of Enthalpy, H Determine Binding affinity, K a Determine Stoichiometry, n Determine Entropy, S 15
09/11/2010 Incremental titration, medium binding 2-CMP (100 µl, 1.6 mm) titrated into RNase A (1.0 ml, 80 µm) 20, 5 µl injections at 25 o C n = 1 K a = 1 x 10 6 M -1 Enthalpy of binding: -65 KJ/mol The shape of the binding curve determines the accuracy of K a. Need curvature. What if K a is outside 10 3 10 9 M -1? Continuous titration 50 BaCl 2 titrated into 18-crown-6 Regular (incremental) ITC experiment: 20-30 data points, 1.5-2 hours Continuous ITC experiment: a thousand data points more precise fit of data curve more accurate K a, n Heat Rate (µw) 45 n = 1.01 40 K a = 5.97 x 10 3 M -1 35 H = -31.4 kj mol -1 30 25 Raw Data 20 Fit 15 10 5 0 200 300 400 500 600 700 800 900 1000 Time (seconds) Continuously inject ligand (0.05-0.15 µl/s) 10 2'-CMP titrated into RNase A 9 20 minute experiment No hardware or software modifications However, binding must be instantaneous Heat Rate (µw) 8 7 6 5 4 3 2 Raw Data Fit n = 1.04 K a = 1.12 x 10 6 M -1 H = -73.4 kj mol -1 1 0 200 300 400 500 600 700 800 900 1000 Time (seconds) 16
09/11/2010 Critical micelle concentration Critical micelle concentration (CMC) is the concentration at which detergents aggregate to form micelles. Titrate concentrated detergent suspension (micelles) into buffer. Initially micelles dissociate in sample cell. At CMC, detergent in the sample cell aggregate. Midpoint of the inflection is the CMC. NanoITC Advantages Precision measurement of (biological) interactions - Heat is a universal detector of all processes NanoITC is the most versatile microcalorimetry instrument available for characterization and analysis of interactions of (biological) molecules In-solution; Native materials - no need to label or immobilize NanoITC generates full thermodynamic profile NanoITC is necessary tool for precision measurements of structure, function, and biomolecular interactions 17
09/11/2010 Nano-DSC Specifically designed to determine thermal stability and heat capacity of proteins and other macromolecules in dilute solution. Unsurpassed assay precision enables maximum data collection in a single experiment. Nano DSC Applications Investigate Protein/Domain structure Determine thermal transition(melting) temperatures Measure H of denaturation Measure reversibility of thermal processes Measure C p of the unfolding process Pre-formulation stability studies Evaluate relative stability of bioengineered proteins Evaluation of high affinity binding (up to 10 20 M -1 ) 18
09/11/2010 DSC Block Diagram DSC Cell Design Continuous Capillary Cylindrical Cell Construction; Inert to biomaterials 99.99% Platinum Small Sample Volume (0.33 ml) Attenuates or delays onset of aggregation until after protein has unfolded Easy-to-fill and clean design Cell Construction; Inert to biomaterials 99.999% Gold Small Sample Volume (0.33 ml) Preferable for high temperature option Competitive Non- Capillary option 19
09/11/2010 Differential Scanning Calorimetry Typical DSC Data 14 T M 12 10 Cp (kcal/mole/ o C) 8 6 4 2 } C p H 0 30 40 50 60 70 80 90 Temperature ( o C) Nano DSC Cell Configuration Typical Non-capillary Cell Aggregation/Precipitation DSC Thermogram Continuous capillary cells delay / inhibit aggregation and precipitation of proteins during scans Aggregation/Precipitation Characteristics Difficult to control Expt parameters important Kinetic event Distortion of peaks inaccurate data 20
09/11/2010 Ligand binding If a ligand binds to a protein, the T m of the protein will increase. Generally, the more bound ligand there is, or the tighter it binds, the more T m increases. Can determine binding constant at T m. Not ideal But, useful if very slow or very tight binding, or organic solvents necessary. Excess Cp / kj mol -1 K -1 80 70 0.3 mm 60 0.15 mm 0.75 mm 50 1 mm 40 1.25 mm 0.075 mm 30 0.05 mm 1.5 mm 20 0 mm 10 0-10 35 45 55 65 75 85 Temperature / C Perfectly valid if comparing relative binding of ligands to same protein DSC is a quick way of screening whether two molecules interact. Membrane protein/membrane interaction About 30% of proteins are associated with membranes. Membranes are hydrophilic outside, hydrophobic inside. Membrane proteins are mostly hydrophobic, difficult to work with, very difficult to purify Does a protein bind to a membrane? Easy to tell by DSC Specific changes in lipid thermogram indicates how protein interacts with membrane (on surface, or internal penetration) Powerful! Most techniques are affected by cloudiness, light scattering. Not DSC! 21
09/11/2010 Membrane protein structure Membrane proteins are very difficult to purify. Micrograms of protein can represent weeks of work. Require detergents for solubilization Using the Nano-DSC, obtained these high-quality scans using 20 µg (0.3 picomoles) of a 70,000 Da complex consisting of 3 membrane proteins Thermogram very well fit by 3 transitions Partial chemical derivatization and stablilization of complex easily verified by DSC Pressure perturbation Pressure perturbation: the heat change in a biopolymer sample caused by a pressure jump. Heat corresponds to the work done by the pressure to create a volume change. Allows calculation of the coefficient of thermal expansion of the biopolymer, which is correlated with hydration of the biopolymer. Volume change can also be correlated with tightness of packing of protein interior (chymotrypsinogen is more hydrophobic than ribonuclease). Nano-DSC can alter pressure quickly and smoothly, in 3 modes. A potentially powerful technique, but very limited interpretable data available. 22
09/11/2010 Summary and outlook The intricate structures of biopolymers are responsible for their activity. The structures are held together by many weak interactions. Proteins are only marginally stable. Understanding the thermodynamics driving biopolymer structure/function lets you design better proteins, design better ligands, understand how biopolymers fold, how they interact with their environment Calorimetry is most direct, versatile and powerful approach for understanding the forces controlling protein structure and function. Grazie per lattenzione Qualche domanda? 23