Metal binding properties of recombinant rat parvalbumin wild-type and F102W mutant.
Rat parvalbumin (PV), an EF-hand type Ca(2+)-binding protein, was expressed 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 recombinant wild-type (PVWT) and F102W mutant (PVF102W) proteins have the expected molecular weight and retain the native structure. Both proteins contain two non-cooperative Ca2+/Mg(2+)-binding sites with intrinsic affinity constants, KCa and KMg, of 2.4 +/- 0.9 x 10(7) M-1 and of 2.9 +/- 0.2 x 10(4) M-1, respectively, for PVWT, and KCa and KMg, of 2.7 +/- 1.1 x 10(7) M-1 and of 4.4 +/- 0.3 x 10(4) M-1, respectively, for PVF102W. Based on the highly similar metal binding properties of PVWT and PVF102W the latter protein was used to study cation-dependent conformational changes. Trp fluorescence emission and UV difference spectra of PVF102W indicated that the Trp residue at position 102 is confined to a hydrophobic core and conformationally strongly restricted. Upon Ca2+ or Mg2+ binding the structural organization of the region around the Trp is hardly affected, but there are significant changes in its electrostatic environment. The conformational change upon binding of Ca2+ and Mg2+, as monitored by UV difference spectrophotometry, increases linearly from 0 to 2 cations bound, indicating that the binding of both ions contributes equally to the structural organization in this protein.
Pauls
T L
TL
Institute of Veterinary Biochemistry, University of Zürich-Irchel, Switzerland.
Durussel
I
I
Cox
J A
JA
Clark
I D
ID
Szabo
A G
AG
Gagné
S M
SM
Sykes
B D
BD
Berchtold
M W
MW
D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008670:Metals / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D010320:Parvalbumins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D008670:Metals; D009838:Oligodeoxyribonucleotides; D010320:Parvalbumins; D011994:Recombinant Proteins; D008274:Magnesium; D002118:Calcium