METAL-BINDING PROPERTIES OF RECOMBINANT RAT PARVALBUMIN WILD-TYPE ANDF102W MUTANT

Rat parvalbumin (PV), an EF-hand type Ca2+-binding protein, was expres sed in Escherichia coli and mutated by replacing a Phe at position 102 with a unique Trp in order to introduce a distinct fluorescent label into the protein. Mass spectroscopy and NMR data indicate that the rec ombinant wild-type (PV(WT)) and F102W mutant (PV(F102W)) proteins have the expected molecular weight and retain the native structure. Both p roteins contain two non-cooperative Ca2+/Mg2+-binding sites with intri nsic affinity constants, K(Ca) and K(Mg), of 2.4 +/- 0.9 x 10(7) M-1 a nd of 2.9 +/- 0.2 x 10(4) M-1, respectively, for PV(WT), and K(Ca) and K(Mg) of 2.7 +/- 1.1 x 10(7) M-1 and of 4.4 +/- 0.3 x 10(4) M-1, resp ectively, for PV(F102W). Based on the highly similar metal binding pro perties of PV(WT) and PV(F102W) the latter protein was used to study c ation-dependent conformational changes. Trp fluorescence emission and UV difference spectra of PV(F102W) indicated that the Trp residue at p osition 102 is confined to a hydrophobic core and conformationally str ongly restricted. Upon Ca2+ or Mg2+ binding the structural organizatio n of the region around the Trp is hardly affected, but there are signi ficant changes in its electrostatic environment. The conformational ch ange upon binding of Ca2+ and Mg2+, as monitored by UV difference spec trophotometry, increases linearly from 0 to 2 cations bound, indicatin g that the binding of both ions contributes equally to the structural organization in this protein.