DSC Characterization of the Structure/Function Relationship for Proteins

Transcription

1 DSC Characterization of the Structure/Function Relationship for Proteins

2 Differential Scanning Calorimetry (DSC) DSC is recognized as Gold Std technique for measuring molecular thermal stability and structure Only technique that gives full thermodynamic profile of molecular structure in one experiment Transition Temperature T m Enthalpy - H Change in Heat Capacity - C p

3 Protein Structure Stability The primary sequence of a protein tells little about the chemistry of the protein. Proteins are only marginally stable. The free energy required to denature a protein is about 0.42 kj/mol of amino acid, so a 100 residue (100 amino acid chain) protein is stabilized by about 42 kj/mol. The energy required to break a single hydrogen bond is about 19 kj/mol - Protein structure is a delicate balance of stabilizing and destabilizing interactions. Interactions with the environment (salts, membranes, ligands, other proteins) are critical to the structure of a protein.

4 Two state model of protein unfolding Heat associated with unfolding (endothermic) and folding (exothermic) is easily measured by calorimetry, allowing thermodynamic analysis of the folding/unfolding process. Folding and unfolding of a small protein, a domain, or a subunit, is cooperative (once started, it goes to completion). These small units can fold and unfold reversibly. Reversibility is directly measurable using DSC.

5 DSC Theory Partial Molar Heat Capacity (kj K -1 mol -1 ) T m C p Temperature ( C) Area = H cal Enthalpic changes (changes in hydrophobic interactions, hydrogen bonding, electrostatic interactions) are compensated by entropic changes (changes in solvation, conformational freedom)

6 What does a DSC scan tell us? Heat capacity change ( C p ; covalent structure and hydration) determined from baseline shift before/after unfolding. Primarily reflects exposure of hydrophobic groups. C p is positive. (Note: 1 W = 1 J/sec. J/sec K/sec = J/K. Normalize for [protein] = C p ) Area under unfolding peak is the enthalpy ( H) of the unfolding reaction. Primarily due to hydrogen bonds breaking, disruption of hydrophobic interactions. Midpoint of the thermal unfolding (T m ): temperature at which half the molecules are unfolded. Indication of the stability of the molecule. DSC is the only technique that allows the direct measure of T m, C p and H. Values are more accurate than indirect (calculated) methods.

7 Instruments

8 Scanning Microcalorimetry Sensitive mw µw nw More Sensitive Discovery DSC MC DSC Nano DSC Diffusion-bonded Sensor 50-Sample Autosampler Sample Size: up to 20 mg Scan Rate: C/min 3 samples 1 reference Sample Flexibility 1 ml sample Vol. Scan Rate: 0-2 C/min 1 sample 1 reference In-solution sample 300 µl active cell Vol. Scan Rate: C/min Automated sample handling

9 Nano DSC Instruments Nano DSC Nano DSC A/S Interface Nano DSC A/S System Nano DSC Nano DSC A/S Interface Nano DSC A/S System Specifically designed for in-solution samples Scan rates: C 2 C/min No labeling or immobilization required True power compensation detection system 96-well plate automation configuration available

10 Nano DSC Design Nano DSC Platinum capillary cells Active cell volume = 300 µl USB connection to computer Innovative sensor design Superior sensitivity

11 Nano DSC Specifications Continuous Capillary Operating temperature: -10 to 130 C Scan rate: to 2 C/min Cell volume: 0.3 ml Cell geometry: Fixed capillary Cell material: Platinum Operating pressure: Up to 6 atmospheres Pressure Perturbation (PPC): Built-in up to 6 atmospheres Response Time: 5 Seconds Heat Measurement Type: Power compensation

12 Easy to Load

13 Nano DSC Autosampler Plumbing Overview The Automated NanoDSC consists of: H 2 O Autosampler with built-in cooling of sample plates Nano DSC - New USB connection to computer Autosampler interface

14 Nano DSC Sensitivity How much protein? 200 HEW Lysozyme in 0.20 M Glycine Buffer, ph 4.0 (2 C/minute) 180 Molar Heat Capacity (kj K -1 mol -1 ) µg µg µg µg µg 100 µg µg Temperature ( C)

15 Benefit of Capillary Sample Cell Figure 1 Figure 2 Data obtained with a DSC with Data obtained with a Nano DSC with Coin Shaped Sample Cell a Continuous Capillary Sample Cell Precipitating protein after unfolding Stable baseline after unfolding heat rate / µj s heat rate / µj s temperature / C temperature / C Purified human IgG 1 monoclonal antibody in physiological buffer; 0.5 mg/ml

16 DSC Applications Biopolymer Stability Absolute Heat Capacities (requires ultra-stable baselines, is required to correct temperature dependency of ITC data Biopolymer Structure Domain and Subunit Organization oligomerization Bio-Engineering Mutant Proteins (improved properties) Ligand Interactions Drug Binding to Proteins or Nucleic Acids Membrane Structure Lipid Bilayers, Membrane proteins Pressure Perturbation Lipid/biopolymer structure and solvation (G, H, C p, C v, α [thermal coefficient of expansion], β [compressibility]) Complex milieu

17 Modeling Data and Thermogram Deconvolution CH2 Fab CH3 Fitting algorithms can successfully and accurately deconvolute this broad, asymmetrical, unfolding event. DSC thermogram (dark blue) fitted to three events (red, green and light blue) Assignment of domains is based on previous data published by Wen in 2008

18 Protein Stability Mutate proteins to make them more stable, more specific, faster, have new properties, etc. Useful to predict outcome of a mutation, so need a database of thermodynamically characterized proteins Complicated network of interactions. Also, enthalpic-entropic compensation Example: enthalpic changes (changes in hydrophobic interactions, hydrogen bonding, electrostatic interactions) compensated by entropic changes (changes in solvation, conformational freedom) Grzesiak et al., J. Mol. Bio.301, , 2000 Since G = H T S, unfolding occurs when T S increases sufficiently (e.g., by absorbing heat) to overcome stabilizing enthalpic interactions Biopolymer unfolding is endothermic BPTI WT = Ala

19 Effect of additives and formulations Proteins used for pharmaceutical or industrial applications require stabilization against chemical and physical degradation Choice of an additive or a formulation (mixture of additives) is generally determined empirically DSC is the fastest way of evaluating additives (effect on T m, reversibility) α-amylase Olsen et al., Thermochimica Acta, 484, (2009), 32-37

20 Liquid Formulation Stability Increases in T m correlate with improved stability of IL-1R DSC data can reliably predict the rank order of stability for various additives Excepient Mole Ratio T m ( o C) Control Sugars Manitol Glucose Polymers / Polyols PEG (300) Ethanol Ethanol Salts NaCl CaCl Surfactants Pluronic F68 Tween Glucose/NaCl 2037 / Remmele, et. al., Pharmaceutical Res. 15, (1998)

21 Studying binding by DSC If a ligand binds preferentially to a folded 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, but not ideal. But, useful if very slow or very tight binding, or organic solvents necessary. (See example) Valid if comparing relative binding of ligands to same protein DSC is a quick way to determine if two molecules interact Excess Cp / kj mol -1 K mm 0.05 mm 0 mm 0.3 mm 0.15 mm Temperature / C 0.75 mm 1 mm 1.25 mm 1.5 mm Binding of 2 -CMP to RNase A ± 5% DMSO K a = 5900 M -1 (-DMSO); 6900 M -1 (+DMSO) at T m NOTE: Using solvent with ITC can be more complex

22 Plasma DSC Profile Courtesy of N. Garbett and J. Chaires University of Louisville

23 N.C. Garbett, J.J. Miller, A.B. Jenson and J.B. Chaires (2008) Calorimetry Outside the Box: A New Window into the Plasma Proteome Biophys. J. 94:

24 Differential Scanning Calorimetry (DSC) DSC is recognized as Gold Std technique for measuring molecular thermal stability and structure Only technique that gives full thermodynamic profile of molecular structure in one experiment Transition Temperature T m Enthalpy - H Change in Heat Capacity - C p

25 Power of ITC & DSC ITC Titration or SI H, K a, n, S DSC Dilute solution Cp, n, S, Tm Structure XRC, AA Seq, Subunits Thermodynamic Profile Inter & Intramolecular characteristics H, K a, n, S, Cp, Tm DSC Subunits & Complexes Cp, Tm, S Corrected TD Profile Binding & unfolding / refolding H, K a, n, S, Cp,Tm Structure Function Relationship Binding sites, Binding mechanisms, Complex stability

26 Thank You