Volume 92, number 2 FEBS LETTERS August 1978 zyxwvuts NON-EQUIVALENCE OF THE CD AND EF SITES OF MUSCULAR PARVALBUMINS. A ’ l 3 Cd NMR STUDY T. DRAKENBERG and B. LINDMAN zyxwvutsrqponmlkjihgfedcbaZYXWVU Division of Physical Chemistry 2, Chemical Center, P.O.B. 740, S-220 07 Lund, Sweden and A. CAVE and J. PARELLO Equipe de Recherche de Biophysique No. 140, C.N.R.S., U.S.T.L., 34060 Montpellier, France Received 26 June 1978 1. Introduction The tertiary structure of parvalbumins is well documented by the determination of atomic coordi- nates for the component ~14.25 from carp muscle by X-ray crystallography [l] . A significant feature of this structure is the presence of two calcium ions at sites named CD and EF respectively [2] . Six oxygen atoms are liganded to the central cation in an octa- hedral arrangement, with Ca . . .O distances ranging from 2.15-2.85 A [l] . The first site contains solely protein ligands: four carboxyl groups, a peptide bond carbonyl group and a serine hydroxyl group. The site EF also contains four carboxyl groups and a carbonyl group but the sixth ligand is provided by a water molecule [l] , This suggests that the parvalbumin sites CD and EF will have different physicochemical and functional properties. Studies of the binding of Ca(I1) by various tech- niques have shown that parvalbumins have two cationic sites with high affinity for calcium ions [3-51, but have not been able to distinguish between the two sites. It has also been shown that many other cations compete for the ionic sites of parvalbumins [6-91. Specific binding of Na(1) [lo] and Mg(II) [ 1 l] has been observed using “Na and “Mg NMR spectroscopy. Kinetic aspects of the binding of Ca(II) to parvalbumins have been investigated directly by 43CaNMR [ 121. Although well adapted to charac- 346 terization of cation binding, these NMR studies have not provided evidence of any nonequivalence of the two ionic sites. As the chemical shifts of ‘r3Cd(II) are very sensitive to the chemical nature of the ligand groups involved in complex formation [ 131, it seemed likely that the CD and EF sites could be distinguished by ‘r3Cd NMR spectroscopy. Calcium and cadmium have very similar ionic radii and are likely to compete for the two sites. It has been shown that the rr3Cd NMR signal from cadmium bound to various proteins can be readily observed [ 14-161. In the present work it is shown that the cadmium ions bound to the carp parvalbumin molecule give rise to two r13CdNMR signals with distinct chemical shifts. It is therefore possible to make competition studies, giving informa- tion for each site separately, by “‘Cd NMR spec- troscopy. This is greatly facilitated by a most favou- rable signal-to-noise ratio as compared with previous reports on ‘13CdNMR of protein solutions. 2. Materials and methods Carp parvalbumin, component pI 4.25 from the muscle of C’y prinus carp@ was used throughout this work. It was prepared according to the procedure of Pechere [ 171 and contained 2.4 calcium ions per molecule (atomic absorption spectrophotometry ElseviwfNorth-Holland Biomedical &ess brought to you by CORE View metadata, citation and similar papers at core.ac.uk provided by Elsevier - Publisher Connector