Effects of fluorescent reporter group structure on the dynamics surrounding cysteine-26 in spinach calmodulin: A model biorecognition element

Conformational changes associated with the Ca²⁺-dependent activation of spinach calmodulin (CaM) have been assessed in aqueous solution by using steady-state and frequency-domain fluorescence spectroscopy of acrylodan-, fluorescein-, and tetramethylrhodamine-labeled CaM. Spinach CaM was site-selectively labeled at cysteine-26 so we could study the dynamics at a well-defined location within the protein. By using multiple fluorophores attached to the same site, we determined the Ca²⁺-dependent changes in the CaM global rotational dynamics, and also determined how the local fluorophore dynamics were affected by the fluorophore polarity, size, and charge. Upon binding Ca²⁺ at pH 7.00, spinach CaM changes its conformation by exposing the acrylodan fluorescent reporter group to a more dipolar environment. There is also a concomitant increase in the fluorescein pKa when Ca²⁺ binds to CaM. The global motions of spinach CaM are described by rotational reorientation times of 8.4 and 10.5 ns for the apo- and Ca²⁺-saturated CaM at 23°C. In light of all the available data in the literature on CaM, these results are consistent with a small expansion of the CaM globular domains, a bending and/or rotation of the central peptide chain that connects the globular domains that host the four Ca²⁺ binding sites (two per domain) in such a way that residues 27 and 139 are brought closer to one another, and/or a difference in the degree of hydration between the apo- and Ca²⁺-saturated CaM.