Calcium-binding and temperature induced transitions in equine lysozyme: new insights from the pCa-temperature "phase diagrams".
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.
Permyakov
Sergei E
SE
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia.
Khokhlova
Tatyana I
TI
Nazipova
Aliya A
AA
Zhadan
Andrey P
AP
Morozova-Roche
Ludmila A
LA
Permyakov
Eugene A
EA
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D004492:Edetic Acid / Q000378:metabolism; D006736:Horses / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007768:Lactalbumin / Q000737:chemistry / Q000378:metabolism; D009113:Muramidase / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
D001059:Apoproteins; D014364:Tryptophan; D007768:Lactalbumin; D004492:Edetic Acid; D009113:Muramidase; D002118:Calcium