One-dimensional 1H NMR was employed to study the effects of Ca2+ and Lu3+ binding on the apo and calcium-saturated forms of dog bone Gla protein (BGP, osteocalcin). Titration of apo dog BGP with Ca2+ in 20 mM NaCl showed spectral perturbations consistent with the binding of 5 mol equiv of calcium in the NMR slow-exchange limit. The first 2 Ca2+ equiv induced significant conformational changes in the apoprotein, binding cooperatively with a Kd1 approximately 5.0 x 10(-4) M and a Hill coefficient H = 2.3 in 20 mM NaCl. The last 3 equiv bound with a slightly weaker affinity and did not induce significant structural changes. Neither the affinity nor the stoichiometry of calcium binding was significantly altered at 150 mM NaCl. The addition of only 1 Lu3+ equiv to apo dog osteocalcin was sufficient to induce the same spectral perturbations as 2 Ca2+ ions. The addition of 2 Lu3+ equiv to calcium-saturated osteocalcin had little effect on its 1H NMR spectrum, and BGP aggregated at [Lu3+]o/[BGP]o ratios greater than 2 in either the presence or absence of calcium. The spectrum of calcium-saturated osteocalcin was invariant at < or = 55 degrees C (< or = 50 degrees C in 150 mM NaCl), after which the proton resonances shifted to frequencies more characteristic of apo BGP. Saturation with calcium somewhat stabilized the apo dog osteocalcin protein against conformational changes induced at pH extremes; apo BGP was stable at 6.0 < or = pH < or = 10, and calcium-saturated BGP was stable at 5.8 < or = pH < or = 10. Both our NMR and gel filtration data indicate that calcium-saturated osteocalcin exists as a dimer at both high and low protein concentrations. A conformational change in dog osteocalcin was thus induced by the cooperative association of Ca2+ to two high-affinity sites on the protein and stabilized by the association of 3 additional Ca2+ equiv. The results of our temperature and calcium binding studies were consistent with an estimated Kd1 approximately 5.0 x 10(-4) M for the two high-affinity sites. Lutetium induced the same structural changes in osteocalcin as calcium, but the two high-affinity Ca2+ binding sites did not have equal affinities for Lu3+. The BGP:Ca2+ complex was unstable at the low pH conditions induced by osteoclasts during bone resorption, yet the osteocalcin protein retained a BGP:Ca(2+)-like conformation at low pH. However, unlike the calcium-saturated form of the protein, osteocalcin was monomeric at low pH.
[NMR paper] Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)
Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin.
Related Articles Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and Cu(II)-rusticyanin.
J Am Chem Soc. 2002 Nov 20;124(46):13698-708
Authors: Donaire A, Jiménez B, Fernández CO, Pierattelli R, Niizeki T, Moratal JM, Hall JF, Kohzuma T, Hasnain SS, Vila AJ
The blue copper proteins (BCPs), pseudoazurin from Achromobacter cycloclastes and rusticyanin from Thiobacillus...
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11-24-2010 08:58 PM
[NMR paper] Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectrosc
Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy.
Related Articles Structure of the metal-water complex in Ras x GDP studied by high-field EPR spectroscopy and 31P NMR spectroscopy.
Biochemistry. 2001 Feb 20;40(7):1884-9
Authors: Rohrer M, Prisner TF, Brügmann O, Käss H, Spoerner M, Wittinghofer A, Kalbitzer HR
The small GTPase Ras plays a key role as a molecular switch in the intercellular signal transduction. On Mg(2+) --> Mn(2+) substituted samples, the first ligand sphere of...
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11-19-2010 08:32 PM
[NMR paper] NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9.
NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9.
Related Articles NMR studies of metal ion binding to the Zn-finger-like HNH motif of colicin E9.
J Inorg Biochem. 2000 Apr;79(1-4):365-70
Authors: Hannan JP, Whittaker SB, Hemmings AM, James R, Kleanthous C, Moore GR
The 134 amino acid DNase domain of colicin E9 contains a zinc-finger-like HNH motif that binds divalent transition metal ions. We have used 1D 1H and 2D 1H-15N NMR methods to characterise the binding of Co2+, Ni2+ and Zn2+ to this protein. Data for the...
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[NMR paper] [13C]Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe
Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.
Related Articles Methionine NMR and metal-binding studies of recombinant human transferrin N-lobe and five methionine mutants: conformational changes and increased sensitivity to chloride.
Biochem J. 1999 Dec 15;344 Pt 3:881-7
Authors: He QY, Mason AB, Tam BM, MacGillivray RT, Woodworth RC
The N-lobe of human serum transferrin (hTF/2N) and single point mutants in which each...
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[NMR paper] Metal ion binding to calmodulin: NMR and fluorescence studies.
Metal ion binding to calmodulin: NMR and fluorescence studies.
Related Articles Metal ion binding to calmodulin: NMR and fluorescence studies.
Biometals. 1998 Sep;11(3):213-22
Authors: Ouyang H, Vogel HJ
Calmodulin is an important second messenger protein which is involved in a large variety of cellular pathways. Calmodulin is sensitive to fluctuations in the intracellular Ca2+ levels and is activated by the binding of four Ca2+ ions. In spite of the important role it plays in signal transduction pathways, it shows a surprisingly broad...
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[NMR paper] The dynamic properties of the M121H azurin metal site as studied by NMR of the parama
The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives.
Related Articles The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives.
J Biol Chem. 1998 Jan 2;273(1):177-85
Authors: Salgado J, Kroes SJ, Berg A, Moratal JM, Canters GW
The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by 1H NMR of the Cu(II) and Co(II) paramagnetic...
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11-17-2010 11:06 PM
[NMR paper] NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.
NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.
Related Articles NMR and stopped-flow studies of metal ion binding to alpha-lactalbumins.
Biochim Biophys Acta. 1996 Mar 7;1293(1):72-82
Authors: Aramini JM, Hiraoki T, Grace MR, Swaddle TW, Chiancone E, Vogel HJ
1H-NMR spectroscopy and stopped-flow techniques have been used to investigate the binding of a host of metal ions to alpha-lactalbumins from bovine, goat, and human sources. We have identified two 1H-NMR markers diagnostic of metal ion binding to the...
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[NMR paper] Mutation of invariant cysteines of mammalian metallothionein alters metal binding cap
Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.
Related Articles Mutation of invariant cysteines of mammalian metallothionein alters metal binding capacity, cadmium resistance, and 113Cd NMR spectrum.
J Biol Chem. 1991 Dec 25;266(36):24390-7
Authors: Cismowski MJ, Narula SS, Armitage IM, Chernaik ML, Huang PC
Using a yeast expression vector system, we have expressed both wild type and six mutated Chinese hamster metallothionein coding sequences in a...