title: Characterization of lanthanide ion binding to the EF-hand protein S100 beta by luminescence spectroscopy.
abstract: S100 beta is a member of a group of low-molecular weight acidic calcium binding proteins widely distributed in the vertebrate nervous system containing two helix-loop-helix calcium binding motifs (sites I and II). In addition, S100 beta also has auxiliary Zn2+ binding sites that are distinct from the Ca2+ binding sites. Luminescence spectroscopy using Eu3+ and Tb3+ as spectroscopic probes for Ca2+ is used to characterize the Ca2+ binding sites of this protein. Eu3+-bound S100 beta shows two distinct Eu3+ binding environments from both the excitation spectrum and Eu3+ excited state lifetimes. Eu3+ bound to the classical EF hand site II has a Kd of 660 +/- 20 nM, whereas the dissociation constant for the pseudo-EF hand site I is significantly weaker. Lifetimes in H2O and D2O lead to the finding that there are four water molecules coordinated to the Eu3+ in the weakly binding site I and two water molecules to the tightly binding site II. Site II in S100 beta expectedly is very similar to high-affinity Ln3+ binding domains I and II in calmodulin. Eu3+ luminescence experiments with Zn2+-loaded S100 beta show that the lifetime for Eu3+ in site I in Zn2+-loaded S100 beta is significantly different than that in the absence of Zn2+. Tyrosine-17-sensitized Tb3+ luminescence experiments indicate that the Tb3+ occupying the proximal weaker binding site I is sensitized, whereas Tb3+ in site II is not. The distance between sites I and II (15.0 +/- 0.4 A) in S100 beta was determined from Forster-type energy transfer in D2O solutions containing bound Eu3+ donor and Nd3+ acceptor ions. For Zn2+-S100 beta, the intersite distance is reduced to 13 +/- 0.3 A. Location of histidine-15 close to pseudo-EF site I suggests that Zn2+ binding likely changes the conformation of this site, causing a reduction of the intersite distance by approximately 2 A.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore 21201, USA.
authors.forename: D
authors.initials: D
authors.lastname: Chaudhuri
authors.forename: W D
authors.initials: WD
authors.lastname: Horrocks
authors.forename: J C
authors.initials: JC
authors.lastname: Amburgey
authors.forename: D J
authors.initials: DJ
authors.lastname: Weber
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004735:Energy Transfer; D005063:Europium / Q000378:metabolism; D008163:Luminescent Measurements; D008674:Metals, Rare Earth / Q000378:metabolism*; D009354:Neodymium / Q000378:metabolism; D009414:Nerve Growth Factors; D011485:Protein Binding; D051381:Rats; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism*; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013725:Terbium / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D008674:Metals, Rare Earth; D009414:Nerve Growth Factors; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D013725:Terbium; D009354:Neodymium; D005063:Europium; D015032:Zinc