Infrared spectroscopy : Method development and ligand binding studies
Abstract: Infrared spectroscopy detects molecular vibrations and assesses the properties of molecules and their environment. It is a powerful technique to detect ligand induced changes in biomolecules as it has distinct signals and provides different levels of structural information. An addition of a dialysis accessory to attenuated total reflection infrared spectroscopy makes this technique more universal for ligand binding studies. It facilitates to study ligand binding of substrates, activators, inhibitors and ions to macromolecules as well as effect of pH, ionic strength or denaturants on the structure of macromolecules, which play an important role in drug development. This method was tested with two proteins cyt c and calcium ATPase. We studied phosphoenol pyruvate (PEP) in different ionization states by infrared spectroscopy combined with theoretical analysis. Theoretical calculations helped to assign the bands. The infrared spectrum of labeled PEP and infrared measurement in D2O also helped in band assignment. We used the method dialysis accessory to attenuated total reflection infrared spectroscopy to investigate the binding of PEP and Mg2+ to pyruvate kinase (PK), where conformational changes of PK were revealed upon binding of PEP and Mg2+. Isotopic labeled PEP helped to assign and evaluate the infrared absorption bands. The difference spectrum of bound and free PEP indicates specific interactions between ligand and protein. The quantitative evaluation revealed that the enzyme environment has little influence on the P-O bond strengths, which are weakened by less than 3% upon binding. The carboxylate absorption bands indicate shortening of the C-O bond by as little as 1.3 pm. The binding of PEP to PK in presence of monovalent cations K+ and Na+ showed that the binding interactions are very similar.
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