Downstream Regulatory Antagonist Modulator Research Articles

Ca2+ and Mg2+ Induced Conformational Changes in Downstream Regulatory Antagonist Modulator (DREAM) Measured by Extrinsic Hydrophobic Probes

DREAM (downstream regulatory element antagonist modulator) is a multifunctional calcium sensor protein involved in regulation of gene expression and K-channel activity in neuronal cells. Here we present spectroscopic studies of the Ca2+ triggered conformational transition in DREAM using the ANS extrinsic hydrophobic probes 1,8-anilinonapthalene and 2,6-anilinonapthalene. Fluorescence emission spectra of the ANS-DREAM complexes show an increase in ANS emission intensity for Ca2+ and Ca2+Mg2+ bound DREAM consistent with an overall decrease in surface polarity due to the Ca2+ binding. From the titration studies the ANS dissociation constants were determined to be: Kd1,8-ANS = 145±15 μM for the apoform and Kd1,8-ANS = 45±2 μM for Ca2+DREAM. Two binding sites for both ANS isomers were identified in apo/Mg2+ and Ca2+/Mg2+Ca2+ protein using fluorescence lifetime measurements. We propose that one binding site is partially solvent exposed and exhibits a shorter lifetime, τ1,8-ANS = 5.8 ns and τ2,6-ANS = 6.8 ns, whereas the second site is buried within the protein matrix with τ1,8-ANS = 16.1 ns and τ2,6-ANS = 10.9 ns. Computational simulation of ligand binding support the two site binding model with one site located between EF-3/4 and the second site near Trp169 and the entering helix of EF-3. Using fluorescence anisotropy decay two correlation times were resolved for apoprotein and Ca2+ bound DREAM. We assign the longer correlation time to a global motion of the protein (Φapo/Mg2+ = 55 ns and ΦCa2+/Mg2+Ca2+ = 26 ns) and the second time to a local motion of the C-terminal domain (Φapo/Mg2+ = 13 ns and ΦCa2+/Mg2+Ca2+ = 6 ns). The 55 ns correlation time is consistent with apo and Mg2+DREAM being in the tetrameric form whereas Ca2+ binding to apo or Mg2+ DREAM promotes formation of DREAM dimer.

Characterization of Conformational Transitions in Dream Protein using Fluorescence Spectroscopy

DREAM (Down Stream Regulatory Antagonist Modulator) belongs to an important class of calcium binding proteins that are involved in transducing the calcium signal into a biological response in neuronal tissue. DREAM is a unique calcium binding protein that directly binds to DNA and regulates the transcription of prodynorphin and c-fos genes in a calcium dependent fashion. Here we report the conformational transitions in DREAM upon calcium binding by monitoring fluorescence properties of tryptophane residue which is located in proximity to the high affinity calcium binding EF-hand 3. Ca2+ association to the protein leads to the hypsochromic shift in the Trp emission spectrum (max = 338nm) compared to the apo form (max = 343 nm) indicating that the calcium induced conformational transition strongly alters fluorescent probe environment. The changes in the Trp emission spectra are strongly influenced by DREAM concentration confirming that alteration of oligomerization state of DREAM can be readily monitored using fluorescence data. The ligand induced changes in the tryptophan environment were further confirmed by the fluorescence quenching studies using iodide and acrydine quenchers. Trp properties were also analyzed using fluorescence lifetime measurements in frequency domain. In both forms, apo- and Ca2+ bond, the tryptophan lifetime can be described using two exponential decay model , with 1 = 1.5 ns and 1=3.5 ns. The Ca2+ association to protein does not significantly alter tryptophan lifetime but increase the fraction of protein with the lifetime of 1.5 ns.