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In JoVE (1)
- Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
Other Publications (6)
Articles by Anthony K. Mittermaier in JoVE
Collecting Variable-concentration Isothermal Titration Calorimetry Datasets in Order to Determine Binding Mechanisms
Lee A. Freiburger1, Anthony K. Mittermaier1, Karine Auclair1
1Department of Chemistry, McGill University
ITC is a powerful tool for studying the binding of a ligand to its host. In complex systems however, several models may fit the data equally well. The method described here provides a means to elucidate the appropriate binding model for complex systems and extract the corresponding thermodynamic parameters.
Other articles by Anthony K. Mittermaier on PubMed
NMR Dynamics-derived Insights into the Binding Properties of a Peptide Interacting with an SH2 Domain
Biochemistry. Jan, 2005 | Pubmed ID: 15641795
The signal transduction protein phospholipase C-gamma1 (PLC-gamma1) is activated when its C-terminal SH2 domain (PLCC) binds the phosphorylated Tyr-1021 site (pTyr-1021) in the beta-platelet-derived growth factor receptor (PDGFR). To better understand the contributions that dynamics make to binding, we have used NMR relaxation experiments to investigate the motional properties of backbone amide and side chain methyl groups in a peptide derived from the pTyr-1021 site of PDGFR, both free and in complex with the PLCC SH2 domain. The free peptide has relaxation properties that are typical for a small, unstructured polymer, while the backbone of the bound peptide is least flexible for residues in the central portion of the binding site with the amplitude of pico- to nanosecond time scale motions increasing toward the C-terminus of the peptide. The increase in large amplitude motion toward the end of the pY1021 peptide is consistent with the bound peptide existing as an ensemble of states with C-terminal residues having the broadest distribution of backbone conformations, while residues in the central binding site are the most restricted. Deuterium spin relaxation experiments establish that the protein-peptide interface is highly dynamic, and this mobility may play an important role in modulating the affinity of the interaction.
Cross-correlated Spin Relaxation Effects in Methyl 1H CPMG-based Relaxation Dispersion Experiments: Complications and a Simple Solution
Journal of Biomolecular NMR. Apr, 2005 | Pubmed ID: 15929000
Artifacts associated with the measurement of methyl (1)H single quantum CPMG-based relaxation dispersion profiles are described. These artifacts arise due to the combination of cross-correlated spin relaxation effects involving intra-methyl (1)H-(1)H dipolar interactions and imperfections in (1)H refocusing pulses that are applied during CPMG intervals that quantify the effects of chemical exchange on measured transverse relaxation rates. As a result substantial errors in extracted exchange parameters can be obtained. A simple 'work-around' is presented where the (1)H chemical shift difference between the exchanging states is extracted from a combination of (13)C single quantum and (13)C-(1)H multiple quantum dispersion profiles. The approach is demonstrated with an application to a folding/unfolding reaction involving a G48M mutant Fyn SH3 domain.
Trends in Biochemical Sciences. Dec, 2009 | Pubmed ID: 19846313
Biological macromolecules are highly flexible and continually undergo conformational fluctuations on a broad spectrum of timescales. It has long been recognized that dynamics have an important role in the action of these molecules. However, the relationship between molecular function and motion is extremely challenging to delineate, because the conformational space available to macromolecules is vast and the relevant excursions can be infrequent and short-lived. Recent advances in solution nuclear magnetic resonance (NMR) spectroscopy permit biomolecular dynamics to be observed with unprecedented detail. Applications of these new NMR techniques to the study of fundamental processes such as binding and catalysis have provided new insights into how living systems operate at an atomic level.
Chembiochem : a European Journal of Chemical Biology. Dec, 2009 | Pubmed ID: 19856370
Journal of Biomolecular NMR. Jul, 2010 | Pubmed ID: 20461447
Cellulose nanocrystals (CNCs) form liquid crystals in aqueous solution that confer alignment to macromolecules and permit the measurement of residual dipolar couplings. CNCs possess many attractive features as an alignment medium. They are inexpensive, non-toxic, chemically inert, and robust to denaturants and temperature. Despite these advantages, CNCs are seldom employed as an alignment medium and the range of their applicability has not yet been explored. We have re-examined the use of CNCs in biomolecular NMR by analyzing the effects concentration, ionic strength, and temperature on molecular alignment. Stable alignment was obtained over wide ranges of temperature (10-70 degrees C) and pH (2.5-8.0), which makes CNCs potentially very useful in studies of thermophilic proteins and acid-stabilized molecules. Notably, we find that CNC suspensions are very sensitive to the concentrations of biological buffers, which must be taken into account when they are used in NMR analyses. These results have led us to develop a general procedure for preparing aligned samples with CNCs. Using the SH3 domain from the Fyn tyrosine kinase as a model system, we find that CNCs produce an alignment frame collinear with that of the commonly used Pf1 bacteriophage alignment medium, but of opposite magnitude.
Nature Structural & Molecular Biology. Mar, 2011 | Pubmed ID: 21278754
Allostery has been studied for many decades, yet it remains challenging to determine experimentally how it occurs at a molecular level. We have developed an approach combining isothermal titration calorimetry, circular dichroism and nuclear magnetic resonance spectroscopy to quantify allostery in terms of protein thermodynamics, structure and dynamics. This strategy was applied to study the interaction between aminoglycoside N-(6')-acetyltransferase-Ii and one of its substrates, acetyl coenzyme A. It was found that homotropic allostery between the two active sites of the homodimeric enzyme is modulated by opposing mechanisms. One follows a classical Koshland-NÃ©methy-Filmer (KNF) paradigm, whereas the other follows a recently proposed mechanism in which partial unfolding of the subunits is coupled to ligand binding. Competition between folding, binding and conformational changes represents a new way to govern energetic communication between binding sites.