Structure, Folding and Dynamics of Proteins and Their Complexes
FEBS Practical Course, Budapest, September 2-9, 2009.
Institute of Enzymology, Hungarian Academy of Sciences
Department of Biochemistry, Eötvös Loránd University
Module THERMO
Module THERMO
Differential scanning microcalorimetry, isothermal titration calorimetry, Thermoflour
(thermal shift) analysis
Key issues / lectures
Differential scanning microcalorimetry
Dmitrii I. Levitsky, A.N. Bach Institute of Biochemistry of Russian Academy of Sciences
1) General principles of the DSC method;
2) Reversible thermal unfolding and revealing of separate calorimetric domains using
computer deconvolution of the DSC curves (examples: various tropomyosin isoforms);
3) Irreversible thermal unfolding, accompanied by protein aggregation measured by
temperature dependences of light scattering (samples: actin, myosin subfragment 1 (S1));
4) Effects of ligands (samples: thermal unfolding of S1 complexes with ADP and Pi
analogs);
5) Thermal unfolding of complicated protein systems (examples: F-actin filaments,
effects of nucleotides, etc.);
6) Protein-protein interactions (samples: complexes of monomeric G-actin with cofilin,
complexes of F-actin with cofilin, myosin heads and tropomyosin);
7) Non-cooperative thermal unfolding of intrinsically disordered proteins (our recent
unpublished results on the thermal unfolding of small heat shock protein Hsp22 as
measured by DSC in combination with other methods)
1
Structure, Folding and Dynamics of Proteins and Their Complexes
FEBS Practical Course, Budapest, September 2-9, 2009.
Institute of Enzymology, Hungarian Academy of Sciences
Department of Biochemistry, Eötvös Loránd University
Module THERMO
Protocol guidelines
Isothermal titration calorimetry and thermal shift assay
Daumantas Matulis, Laboratory of Biothermodynamics and Drug Design, Institute of
Biotechnology, Vilnius
Demonstration of the measurement of a protein-ligand binding thermodynamic
parameters by isothermal titration calorimetry (ITC) and thermal shift assay (TSA). ITC
enables the determination of the binding constant, binding enthalpy, and binding entropy
in a single experiment. TSA determines the binding constant and the enthalpy of protein
unfolding. TSA consumes about 10 times less material and provides additional
information on protein stability characterization.
Materials used:
Carbonic anhydrase I, Sigma C4396
Methazolamide, Sigma M4156
Sypro Orange dye, Molecular Probes
Standard buffers and salts
Equipment needed:
ITC200, Microcal Inc., or Nano ITC-III, Calorimetry Sciences Corp.
pH-meter, spectrophotometer, balance.
Students are encouraged to bring their own samples. We will bring these materials except
Sypro Orange and buffers and salts.
2
Structure, Folding and Dynamics of Proteins and Their Complexes
FEBS Practical Course, Budapest, September 2-9, 2009.
Institute of Enzymology, Hungarian Academy of Sciences
Department of Biochemistry, Eötvös Loránd University
Module THERMO
Reading list
DSC
Sturtevant, J.
Biochemical applications of differential scanning calorimetry.
Ann. Rev. Phys. Chem. 1987. 38:463-88
Privalov PL, Dragan AI.
Microcalorimetry of biological macromolecules.
Biophys Chem. 2007 Mar;126(1-3):16-24. Epub 2006 May 13. Review. PubMed PMID:
16781052.
Thermoflour/thermal shift
Ericsson UB, Hallberg BM, Detitta GT, Dekker N, Nordlund P.
Thermofluor-based high-throughput stability optimization of proteins for structural
studies.
Anal Biochem. 2006 Oct 15;357(2):289-98. Epub 2006 Aug 10. PubMed PMID:
16962548.
Zubrienė A, Matulienė J, Baranauskienė L, Jachno J, Torresan J, Michailovienė V,
Cimmperman P, Matulis D.
Measurement of Nanomolar Dissociation Constants byTitration Calorimetry and Thermal
Shift Assay - Radicicol Binding to Hsp90 and Ethoxzolamide Binding to CAII.
Int J Mol Sci. 2009 Jun 10;10(6):2662-2680. PubMed
PMID: 19582223; PubMed Central PMCID: PMC2705510.
3