Crystal structure of mouse mu‐crystallin complexed with NADPH and the T3 thyroid hormone
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Mu-crystallin (CRYM), first described as a structural component of the eye lens in marsupials, has been characterized as an NADPH-dependent cytosolic T3 thyroid hormone (triiodothyronine) binding protein. More recently, CRYM has also been associated with ketimine reductase activity. Here, we report three crystal structures: mouse CRYM (mCRYM) in its apo form, in a form complexed with NADPH, and in a form with both NADPH and triiodothyronine bound. Comparison of the apo and NADPH forms reveals a rearrangement of the protein upon NADPH binding that reduces the degrees of freedom of several residues and traps the conformation of the binding pocket in a more T3 competent state. These findings are in agreement with the cooperative mechanism identified using isothermal titration calorimetry. Our structure with T3 reveals for the first time the location of the hormone binding site and shows its detailed interactions. T3 binding involves mainly hydrophobic interactions. Only five residues, either directly or through bridging water molecules, are hydrogen bonded to the hormone. Using in silico docking analysis, a series of ring-containing hydrophobic molecules were identified as potential mCRYM ligands, suggesting that the specificity for the recognition of the hydrophobic part of the hormone might be low. This is in agreement with the ketimine reductase activity that has been identified for ovine CRYM, as it demonstrates how a protein known as a thyroid hormone transporter can accommodate the ringed molecules required for its ketimine reductase activity. In the light of our results, a putative role of CRYM in thyroid hormone metabolism is also discussed.CRYM and CRYM bind by x-ray crystallography (View interaction).Keywords:
Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry
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The interaction between Urinary Trypsin Inhibitor(UTI) and 1-anilino-8-naphthalisene Sulfonate(ANS) was investigated by fluorescence spectrum,Isothermal Titration Calorimetry(ITC) and molecular model.The results of the three experiments identically revealed the presence of four specific binding sites on UTI for ANS,and the interaction between UTI and ANS in the four specific binding sites were driven mainly by electrostatic interaction.The four binding sites were named as site Ⅰ,Ⅱ,Ⅲ and Ⅳ respectively.Site Ⅰ was located in domain Ⅱ and near to Trp98;site Ⅱ were located in the interaction region of the two domains;site Ⅲ and site Ⅳ were located in domain Ⅰ.The results indicated there were four hydrophobic patches in UTI.But the data of ITC experiments showed the presence of other five nonspecific binding sites.The interaction between UTI and ANS in the five nonspecific binding sites were driven mainly by the formation of salt band between the sulfonates of ANS and positive residues on the surface of UTI,which indicated there were five positive residues in the surface of UTI molecular in the neutral buffer.
Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry
Isothermal microcalorimetry
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Stoichiometry
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Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry
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Isothermal Titration Calorimetry
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Isothermal titration calorimetry (ITC) is now an established and invaluable method for determining the thermodynamic constants, association constant and stoichiometry of molecular interactions in aqueous solutions. The technique has become widely used by biochemists to study protein interaction with other proteins, small molecules, metal ions, lipids, nucleic acids and carbohydrates; and nucleic acid interaction with small molecules. The drug discovery industry has utilized this approach to measure protein (or nucleic acid) interaction with drug candidates. ITC has been used to screen candidates, guide the design of potential drugs and validate the modelling used in structure-based drug design. Emerging disciplines including nanotechnology and drug delivery could benefit greatly from ITC in enhancing their understanding and control of nano-particle assembly, and drug binding and controlled release.
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Techniques such as calorimetry, spectroscopy, and hydrodynamic methods can be used to investigate the binding energetics of drugs bound to macromolecules. In this chapter, the authors describe the use of isothermal titration calorimetry (ITC) to measure the binding energetics of drugs bound to blood proteins (i.e., human serum albumin [HSA] and α-acid glycoprotein [AGP]). The stoichiometry (n), the association-binding constant (K a), and the enthalpy (ΔH 0) of binding can be rapidly, directly, and precisely measured using ITC. Because the free energy (ΔG 0) and the entropy (ΔS 0) are readily calculated from K a and ΔH 0, a complete thermodynamic characterization of binding can be acquired in a single experiment.
Isothermal Titration Calorimetry
Energetics
Binding constant
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