Ion-binding properties of recombinant S100beta and two derivatives with either an inactivated Ca2+ site II or a normalized Ca2+ site I.
S100beta contains one unusual and one canonical Ca2+-binding motif. In this study, we measured Ca2+-binding and ensuing conformational changes of recombinant S100beta (rS100beta) and of two mutant forms in which either the canonical loop was inactivated (NoEF) or the unusual one replaced by a canonical one (Caloops). Caloops binds two Ca2+ per monomer with a 3-fold higher affinity than rS100beta; the affinity of NoEF was too low for accurate direct determination. All three proteins bind 3-4 Zn2+ per monomer. Tyrosine 17 fluorescence spectra showed a decrease of intensity upon binding of Ca2+ to the three proteins and an increase upon binding of Zn2+ to rS100beta and NoEF but not in Caloops. The fluorescence change as a function of the Ca2+ concentration yielded half-maximal changes ([Ca2+]0.5) at 1.7, 11.3 and 0.55 mM free Ca2+ for rS100beta NoEF and Caloops, respectively. Our data demonstrate that in S100beta alterations in one site can affect the Ca2+ binding properties of the other site.
Durussel
I
I
Department of Biochemistry, University of Geneva, Switzerland.
Van Eldik
L J
LJ
Cox
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D019281:Dimerization; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics; D009414:Nerve Growth Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009457:Neuroglia / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D017421:Sequence Analysis; D013050:Spectrometry, Fluorescence; D014443:Tyrosine / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D002135:Calcium-Binding Proteins; D009414:Nerve Growth Factors; D011994:Recombinant Proteins; D064568:S100 Calcium Binding Protein beta Subunit; D009418:S100 Proteins; D014443:Tyrosine; D015032:Zinc; D002118:Calcium