The Use of the Free Metal – Temperature ‘Phase Diagrams’ for Studies of Single Site Metal Binding Proteins
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Typical physico-chemical studies of metal binding proteins are usually aimed at determination of the metal binding constant K for a native protein (K n), while the significance of the K value for the thermally denatured protein (K u) is usually underestimated. Meanwhile, metal binding induced shift of thermal denaturation transition of a single site metal binding protein is defined by K n to K u ratio, implying that knowledge of both K values is required for full characterization of the system. In the present work, the most universal approach to the studies of single site metal binding proteins, namely construction of a protein “phase diagram” in coordinates of free metal ion concentration – temperature, is considered in detail. The detailed algorithm of construction of the phase diagrams along with underlying mathematic procedures developed here may be of use for studies of other simple protein-target type systems, where target represents low molecular weight ligand. Analysis of the simplest protein-ligand system reveals that thermodynamic properties of apo-protein dictate the maximal possible increase of its affinity to any simple ligand upon thermal denaturation of the protein. Experimental and general problems coupled with the use of the phase diagrams are discussed.
Keywordsligand binding metal binding phase transition stability thermodynamics
We are indebted to Prof. R. Kretsinger and Dr. V.N. Uversky for very useful discussion of our work and correction of the manuscript. This work was supported by grants from the Programs of the Russian Academy of Sciences (“Fundamental science for medicine” and “Molecular and cellular biology”), Grant 02.442.11.7542 of the Russian Federal Agency for Science and Innovations, Grant 04-04-97322 of the Russian Fund for Basic Research and individual grants to S.E.P. from the “Russian Science Support Foundation”, RUB2-010001-PU-05 CRDF and the Visby Programme of the Swedish Institute.