Formation of monomeric S100B and S100A11 proteins at low ionic strength.

The S100 proteins comprise a group of EF-hand proteins that undergo a calcium-induced conformational change allowing them to interact with other proteins and produce a biological response. A unique feature of these proteins is the fact that they can form both homo- and heterodimers independent of calcium binding. The reported dissociation constants for several S100 proteins span a very large range, from 1-4 microM to <<1 nM, suggesting that differing interface surface areas could govern the strength of the binding affinity. In this work, we examine the dimerization mechanism of S100B and S100A11 in the absence of calcium. Using electrospray mass spectrometry, we demonstrate that the monomer-dimer equilibrium in these S100 proteins is strongly dependent on the ionic strength of the solution. At higher ionic strengths (>or=22 mM), both S100A11 and S100B exist predominantly as homodimers. For apo-S100A11, a K(dimer) near 0.01 microM is estimated, while concentration-dependent experiments under these conditions show the K(dimer) for apo-S100B must be even lower. In contrast, lowering the ionic strength results in the formation of monomeric proteins with poorer dimer propensity. For example, the estimated K(dimer) for apo-S100A11 is more than 400 microM at 0.1 mM NH(4)Ac. (1)H-(15)N HSQC NMR experiments in combination with circular dichroism studies show that monomeric S100B and S100A11 proteins are alpha-helical and retain a significant amount of tertiary structure. Our results indicate that apo-S100B has at least a 10-fold stronger propensity to form dimers than does apo-S100A11 in line with a 400 A(2) greater buried surface area for apo-S100B at its dimer interface. These experiments are the first to show that folded monomeric S100 proteins can be isolated, thus paving the way for future experiments aimed at examining the possible role of these monomers in folding and calcium signaling.