H-1, C-13 AND N-15 NMR ASSIGNMENTS AND SOLUTION SECONDARY STRUCTURE OF RAT APO-S100-BETA

The H-1, C-13 and N-15 NMR assignments of the backbone and side-chain resonances of rat S100 beta were made at pH 6.5 and 37 degrees C using heteronuclear multidimensional NMR spectroscopy. Analysis of the NOE correlations, together with amide exchange rate and H-1(alpha), C-13(alpha) and C-13(beta) chemical shift data, provided extensive secondary structural information. Thus, the secondary structure of S100 beta was determined to comprise four helices (Leu(3)-Ser(18), helix I; Lys(29)-Leu(40), helix II; Gln(50)-Glu(62), helix III; and Phe(70)-Ala(83), helix IV), four loops (Gly(19)-His(25), loop I; Ser(41)-Glu(49), loop II; Asp(63)-Gly(66), loop III; and Cys(84)-Glu(91), loop IV) and two beta-strands (Lys(26)-Lys(28), beta-strand I and Glu(67)-Asp(69), beta-strand II). The beta-strands were found to align in an antiparallel manner to form a very small beta-sheet. This secondary structure is consistent with predictions that S100 beta contains two 'helix-loop-helix' Ca2+-binding motifs known as EF-hands. The alignment of the beta-sheet, which brings the two EF-hand domains of S100 beta into close proximity, is similar to that of several other Ca2+ ion-binding proteins.