ION-BINDING PROPERTIES OF RECOMBINANT S100-BETA AND 2 DERIVATIVES WITH EITHER AN INACTIVATED CA2-II OR A NORMALIZED CA2+ SITE-I( SITE)

S100 beta contains one unusual and one canonical Ca2+-binding motif. I n this study, we measured Ca2+-binding and ensuing conformational chan ges of recombinant S100 beta (rS100 beta) and of two mutant forms in w hich either the canonical loop was inactivated (NoEF) or the unusual o ne replaced by a canonical one (Caloops). Caloops binds two Ca2+ per m onomer with a 3-fold higher affinity than rS100 beta; 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 a n increase upon binding of Zn2+ to rS100 beta and NoEF but not in Calo ops. The fluorescence change as a function of the Ca2+ concentration y ielded half-maximal changes ([Ca2+](0.5)) at 1.7, 11.3 and 0.55 mM fre e Ca2+ for rS100 beta, NoEF and Caloops, respectively. Our data demons trate that in S100 beta alterations in one site can affect the Ca2+ bi nding properties of the other site. (C) 1997 Elsevier Science B.V.