Metal binding properties of recombinant rat parvalbumin wild-type and F102W mutant

• Published in: The Journal of biological chemistry Vol. 268; no. 28; pp. 20897 - 20903
• Main Authors: PAULS, T. L, DURUSSEL, I, COX, J. A, CLARK, I. D, SZABO, A. G, GAGNE, S. M, SYKES, B. D, BERCHTOLD, M. W
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.10.1993
• Subjects: Analytical, structural and metabolic biochemistry; Animals; Base Sequence; Binding and carrier proteins; Biological and medical sciences; Calcium - metabolism; Fundamental and applied biological sciences. Psychology; Magnesium - metabolism; Magnetic Resonance Spectroscopy; Mass Spectrometry; Metals - metabolism; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Parvalbumins - chemistry; Parvalbumins - genetics; Parvalbumins - metabolism; Proteins; Rats; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Binding protein; Ligand binding; Vertebrata; Mammalia; Calcium; Structural unit; Rat; Rodentia; Magnesium; Parvalbumin; Conformation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)36871-1

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.