kinITC: a new method for obtaining joint thermodynamic and kinetic data by isothermal titration calorimetry.

Isothermal titration calorimetry (ITC) is the method of choice for obtaining thermodynamic data on a great variety of systems. Here we show that modern ITC apparatus and new processing methods allow researchers to obtain a complete kinetic description of systems more diverse than previously thought, ranging from simple ligand binding to complex RNA folding. We illustrate these new features with a simple case (HIV-1 reverse transcriptase/inhibitor interaction) and with the more complex case of the folding of a riboswitch triggered by the binding of its ligand. The originality of the new kinITC method lies in its ability to dissect, both thermodynamically and kinetically, the two components: primary ligand binding and subsequent RNA folding. We are not aware of another single method that can yield, in a simple way, such deep insight into a composite process. Our study also rationalizes common observations from daily ITC use.

[1]  A. Ferré-D’Amaré,et al.  Thermodynamic analysis of ligand binding and ligand binding-induced tertiary structure formation by the thiamine pyrophosphate riboswitch. , 2010, RNA.

[2]  M. L. Bianconi,et al.  Calorimetric Determination of Thermodynamic Parameters of Reaction Reveals Different Enthalpic Compensations of the Yeast Hexokinase Isozymes* , 2003, Journal of Biological Chemistry.

[3]  T. Kawasaki,et al.  Thiamine uptake in Escherichia coli. I. General properties of thiamine uptake system in Escherichia coli. , 1969, Archives of biochemistry and biophysics.

[4]  A. Serganov,et al.  Structural basis for gene regulation by a thiamine pyrophosphate-sensing riboswitch , 2006, Nature.

[5]  M. Radi,et al.  Discovery of chiral cyclopropyl dihydro-alkylthio-benzyl-oxopyrimidine (S-DABO) derivatives as potent HIV-1 reverse transcriptase inhibitors with high activity against clinically relevant mutants. , 2009, Journal of medicinal chemistry.

[6]  Keiji Takamoto,et al.  Semi-automated, single-band peak-fitting analysis of hydroxyl radical nucleic acid footprint autoradiograms for the quantitative analysis of transitions. , 2004, Nucleic acids research.

[7]  M. Brenowitz,et al.  Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins , 2006, Nucleic acids research.

[8]  J. Adams,et al.  Inhibition of HIV-1 replication by a nonnucleoside reverse transcriptase inhibitor. , 1990, Science.

[9]  D. Crothers,et al.  The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. , 2005, Molecular cell.

[10]  M. L. Bianconi,et al.  Calorimetry of enzyme-catalyzed reactions. , 2007, Biophysical chemistry.

[11]  A. Cooper,et al.  Heat capacity effects in protein folding and ligand binding: a re-evaluation of the role of water in biomolecular thermodynamics. , 2005, Biophysical chemistry.

[12]  D. Stuart,et al.  High resolution structures of HIV-1 RT from four RT-inhibitor complexes. , 1996 .

[13]  N. Ban,et al.  Structure of the Eukaryotic Thiamine Pyrophosphate Riboswitch with Its Regulatory Ligand , 2006, Science.

[14]  P. Roach,et al.  Thiamine Biosynthesis in Escherichia coli , 2004, Journal of Biological Chemistry.

[15]  M. Sano,et al.  Genetic regulation mediated by thiamin pyrophosphate‐binding motif in Saccharomyces cerevisiae , 2005, Molecular microbiology.

[16]  E. Freire,et al.  Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. , 1991, Biochemistry.

[17]  M. Radi,et al.  Crystal structure of HIV-1 reverse transcriptase bound to a non-nucleoside inhibitor with a novel mechanism of action. , 2010, Angewandte Chemie.

[18]  A. Feig,et al.  Heat capacity changes associated with nucleic acid folding , 2006, Biopolymers.

[19]  R. Georgiadis,et al.  Kinetic discrimination of sequence-specific DNA-drug binding measured by surface plasmon resonance imaging and comparison to solution-phase measurements. , 2007, Journal of the American Chemical Society.

[20]  Renato Zenobi,et al.  Label‐free determination of protein–ligand binding constants using mass spectrometry and validation using surface plasmon resonance and isothermal titration calorimetry , 2009, Journal of molecular recognition : JMR.

[21]  Daniel Herschlag,et al.  Semiautomated and rapid quantification of nucleic acid footprinting and structure mapping experiments , 2008, Nature Protocols.

[22]  U Helena Danielson,et al.  Interaction kinetic characterization of HIV-1 reverse transcriptase non-nucleoside inhibitor resistance. , 2006, Journal of medicinal chemistry.

[23]  J. Chaires,et al.  Calorimetry and thermodynamics in drug design. , 2008, Annual review of biophysics.

[24]  A. Velázquez‐Campoy,et al.  Isothermal titration calorimetry , 1990 .

[25]  D. Crothers,et al.  The kinetics of ligand binding by an adenine-sensing riboswitch. , 2005, Biochemistry.

[26]  M. Gelfand,et al.  Comparative Genomics of Thiamin Biosynthesis in Procaryotes , 2002, The Journal of Biological Chemistry.

[27]  I. Jelesarov,et al.  Survey of the year 2008: applications of isothermal titration calorimetry , 2010, Journal of molecular recognition : JMR.

[28]  T. Tullius,et al.  DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[29]  R. Altman,et al.  SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. , 2005, RNA.

[30]  Ronald R. Breaker,et al.  Thiamine derivatives bind messenger RNAs directly to regulate bacterial gene expression , 2002, Nature.

[31]  Heiko Zettl,et al.  Comparative thermodynamic analysis of DNA--protein interactions using surface plasmon resonance and fluorescence correlation spectroscopy. , 2003, Biochemistry.