ABSTRACT
Isothermal titration calorimetry (ITC) is a label free technique used for direct detection of biological interactions by measurement of the heat given off or taken up during the reaction. In this article we will introduce the ITC technique and review two applications of ITC in drug discovery; small molecule/protein interactions and enzyme kinetics. We will also describe the characteristics of a new miniaturized, ultrasensitive calorimeter. This new microcalorimetry system reduces the quantity of protein (or other macromolecule sample) required to obtain a complete thermodynamic profile (n, K, DeltaH and DeltaS) by up to 7-fold. The reduction in required sample quantities allows ITC to be effectively utilized at earlier stages of the drug discovery and development process.
Subject(s)
Calorimetry/methods , Calorimetry/instrumentation , Carbonic Anhydrase II/antagonists & inhibitors , Carbonic Anhydrase II/metabolism , Drug Discovery/instrumentation , Drug Discovery/methods , Electrochemistry , Enzyme Inhibitors/analysis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Kinetics , Ligands , Ribonucleases/antagonists & inhibitors , Ribonucleases/metabolism , Staining and Labeling , Structure-Activity Relationship , ThermodynamicsABSTRACT
Sac7d is a small chromatin protein from the hyperthermophile Sulfolobus acidocaldarius which kinks duplex DNA by approximately 66 degrees at a single base pair step with intercalation of V26 and M29 side chains. Site-directed mutagenesis coupled with calorimetric and spectroscopic data has been used to characterize the influence of the intercalating side chains on the structure and thermodynamics of the DNA complex from 5 to 85 degrees C. Two single-alanine substitutions (V26A and M29A) and five double-glycine, -alanine, -leucine, -phenylalanine, and -tryptophan substitutions of the surface residues have been created. NMR and fluorescence titrations indicated that the substitutions had little effect on the structure of the protein or DNA binding site size. Each of the mutant proteins demonstrated a temperature-dependent binding enthalpy which was correlated with a similar temperature dependence in the structure of the complex reflected by changes in fluorescence and circular dichroism. A positive heat capacity change (DeltaC(p)) for DNA binding was observed for only those mutants which also demonstrated a thermotropic structural transition in the complex, and the temperature range for the positive DeltaC(p) coincided with that observed for the structural transition. The thermodynamic data are interpreted using a model in which binding is linked to an endothermic distortion of the DNA in the complex. The results support the proposal that the unfavorable enthalpy of binding of Sac7d at 25 degrees C is due in part to the distortion of DNA.
Subject(s)
Archaeal Proteins/chemistry , Archaeal Proteins/genetics , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA/chemistry , DNA/metabolism , Intercalating Agents/metabolism , Nucleic Acid Conformation , Temperature , Thermodynamics , Binding Sites/genetics , Calorimetry , Circular Dichroism , Intercalating Agents/chemistry , Methionine/genetics , Mutagenesis, Site-Directed , Nucleic Acid Heteroduplexes/chemistry , Nucleic Acid Heteroduplexes/metabolism , Protein Binding/genetics , Protein Structure, Secondary , Spectrometry, Fluorescence , Sulfolobus acidocaldarius/chemistry , Sulfolobus acidocaldarius/genetics , Valine/geneticsABSTRACT
Sac7d is a hyperthermophile chromatin protein which binds non-specifically to the minor groove of duplex DNA and induces a sharp kink of 66 degrees with intercalation of valine and methionine side-chains. We have utilized the thermal stability of Sac7d and the lack of sequence specificity to define the thermodynamics of DNA binding over a wide temperature range. The binding affinity for poly(dGdC) was moderate at 25 degrees C (Ka = 3.5(+/-1.6) x 10(6) M(-1)) and increased by nearly an order of magnitude from 10 degrees C to 80 degrees C. The enthalpy of binding was unfavorable at 25 degrees C, and decreased linearly from 5 degrees C to 60 degrees C. A positive binding heat at 25 degrees C is attributed in part to the energy of distorting DNA, and ensures that the temperature of maximal binding affinity (75.1+/-5.6 degrees C) is near the growth temperature of Sulfolobus acidocaldarius. Truncation of the two intercalating residues to alanine led to a decreased ability to bend and unwind DNA at 25 degrees C with a small decrease in binding affinity. The energy gained from intercalation is slightly greater than the free energy penalty of bending duplex DNA. Surprisingly, reduced distortion from the double alanine substitution did not lead to a significant decrease in the heat of binding at 25 degrees C. In addition, an anomalous positive DeltaCp of binding was observed for the double alanine mutant protein which could not be explained by the change in polar and apolar accessible surface areas. Both the larger than expected binding enthalpy and the positive heat capacity can be explained by a temperature dependent structural transition in the protein-DNA complex with a Tm of 15-20 degrees C and a DeltaH of 15 kcal/mol. Data are discussed which indicate that the endothermic transition in the complex is consistent with DNA distortion.
