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 o f spinach calmodulin (CaM) have been assessed in aqueous solution by u sing steady-state and frequency-domain fluorescence spectroscopy of ac rylodan-, fluorescein-, and tetramethylrhodamine-labeled CaM. Spinach CaM was site-selectively labeled at cysteine-26 se we could study the dynamics at a well-defined location within the protein. By using multi ple fluorophores attached to the same site, we determined the Ca2+-dep endent changes in the CaM global rotational dynamics, and also determi ned how the local fluorophore dynamics were affected by the fluorophor e polarity, size, and charge. Upon binding Ca2+ at pH 7.00, spinach Ca M changes its conformation by exposing the acrylodan fluorescent repor ter group to a more dipolar environment. There is also a concomitant i ncrease in the fluorescein pKa when Ca2+ binds to CaM. The global moti ons 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 degrees C. I n light of all the available data in the literature on CaM, these resu lts 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 (tno per do main) 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 a po- and Ca2+-saturated CaM.