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Author  |
Permyakov, S.E.; Khokhlova, T.I.; Nazipova, A.A.; Zhadan, A.P.; Morozova-Roche, L.A.; Permyakov, E.A. |
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Title |
Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature “phase diagrams” |
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Journal Article |
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Year |
2006 |
Publication |
Proteins |
Abbreviated Journal |
Proteins |
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Volume |
65 |
Issue |
4 |
Pages |
984-998 |
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Keywords |
Animals; Apoproteins/chemistry/metabolism; Binding Sites; Calcium/chemistry/*metabolism; Cattle; Edetic Acid/metabolism; Horses/metabolism; Hydrogen-Ion Concentration; Lactalbumin/chemistry/metabolism; Muramidase/*chemistry/*metabolism; Protein Denaturation; Spectrometry, Fluorescence; *Temperature; Thermodynamics; Tryptophan/chemistry/metabolism |
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Abstract |
The most universal approach to the studies of metal binding properties of single-site metal binding proteins, i.e., construction of a “phase diagram” in coordinates of free metal ion concentration-temperature, has been applied to equine lysozyme (EQL). EQL has one relatively strong calcium binding site and shows two thermal transitions, but only one of them is Ca(2+)-dependent. It has been found that the Ca(2+)-dependent behavior of the low temperature thermal transition (I) of EQL can be adequately described based upon the simplest four-states scheme of metal- and temperature-induced structural changes in a protein. All thermodynamic parameters of this scheme were determined experimentally and used for construction of the EQL phase diagram in the pCa-temperature space. Comparison of the phase diagram with that for alpha-lactalbumin (alpha-LA), a close homologue of lysozyme, allows visualization of the differences in thermodynamic behavior of the two proteins. The thermal stability of apo-EQL (transition I) closely resembles that for apo-alpha-LA (mid-temperature 25 degrees C), while the thermal stabilities of their Ca(2+)-bound forms are almost indistinguishable. The native state of EQL has three orders of magnitude lower affinity for Ca(2+) in comparison with alpha-LA while its thermally unfolded state (after the I transition) has about one order lower (K = 15M(-1)) affinity for calcium. Circular dichroism studies of the apo-lysozyme state after the first thermal transition show that it shares common features with the molten globule state of alpha-LA. |
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Address |
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia |
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Language |
English |
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ISSN |
1097-0134 |
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Notes |
PMID:17022083 |
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1858 |
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