Engineering competitive magnesium binding into the first EF-hand of skeletal troponin C

Abstract

The goal of this study was to examine the mechanism of magnesium binding to the regulatory domain of skeletal troponin C (TnC). The fluorescence of Trp29, immediately preceding the first calcium-binding loop in TnCF29W, was unchanged by addition of magnesium, but increased upon calcium binding with an affinity of 3.3 μM. However, the calcium-dependent increase in TnCF29W fluorescence could be reversed by addition of magnesium, with a calculated competitive magnesium affinity of 2.2 mM. When a Z acid pair was introduced into the first EF-hand of TnCF29W, the fluorescence of G34DTnCF29W increased upon addition of magnesium or calcium with affinities of 295 and 1.9 μM, respectively. Addition of 3 mM magnesium decreased the calcium sensitivity of TnCF29W and G34DTnCF29W ∼2- and 6-fold, respectively. Exchange of G34DTnCF29W into skinned psoas muscle fibers decreased fiber calcium sensitivity ∼1.7-fold compared with TnCF29W at 1 mM [magnesium]free and ∼3.2-fold at 3 mM [magnesium]free. Thus, incorporation of a Z acid pair into the first EF-hand allows it to bind magnesium with high affinity. Furthermore, the data suggests that the second EF-hand, but not the first, of TnC is responsible for the competitive magnesium binding to the regulatory domain.