Downstream Regulatory Element Antagonist Modulator (DREAM) belongs to the family of neuronal calcium sensors (NCS) that transduce the intracellular changes in Ca 21 concentration into a variety of responses including gene expression, regulation of Kv channel activity, and calcium homeostasis. Despite the significant sequence and structural similarities with other NCS members, DREAM shows several features unique among NCS such as formation of a tetramer in the apo-state, and interactions with various intracellular biomacromolecules including DNA, presenilin, Kv channels, and calmodulin. Here we use spectroscopic techniques in combination with molecular dynamics simulation to study conformational changes induced by Ca 21 /Mg 21 association to DREAM. Our data indicate a minor impact of Ca 21 association on the overall structure of the Nand C-terminal domains, although Ca 21 binding decreases the conformational heterogeneity as evident from the decrease in the fluorescence lifetime distribution in the Ca 21 bound forms of the protein. Time-resolved fluorescence data indicate that Ca 21 binding triggers a conformational transition that is characterized by more efficient quenching of Trp residue. The unfolding of DREAM occurs through an partially unfolded intermediate that is stabilized by Ca 21 association to EF-hand 3 and EF-hand 4. The native state is stabilized with respect to the partially unfolded state only in the presence of both Ca 21 and Mg 21 suggesting that, under physiological conditions, Ca 21 free DREAM exhibits a high conformational flexibility that may facilitate its physiological functions.
Photoactivable bioactive molecules, often termed "caged" compounds, have attracted significant attention as useful tools for photo-regulating enzymatic activity. Here we examine the mechanism associated with photo-release of urea from a caged urea compound, N-(2-nitrobenzyl)urea, using photothermal beam deflection and time-resolved absorption spectroscopy. Photodissociation of the caged urea results in the prompt formation of an aci-nitro intermediate that decays to nitrosobenzaldehyde by releasing urea with the rate constant of 4.5x10(3) s(-1). Appearance of the aci-nitro intermediate is associated with a volume contraction of -13+/-1 mL mol(-1) and a negligible change in enthalpy (DeltaH=6+/-4 kcal mol(-1)). On the microsecond time-scale, the conversion of the aci-nitro intermediate and concomitant release of urea result in a volume expansion of 6+/-2 mL mol(-1) and a negative enthalpy change of -25+/-5 kcal mol(-1). No additional processes were observed on the timescale up to 100 ms suggesting that the breakdown of the aci-nitro intermediate is the rate-limiting step for urea photo-release. These results suggest a similar mechanism for caged urea photo release as determined previously for the caged ATP compound.
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. Ca 2þ association to the protein leads to the hypsochromic shift in the Trp emission spectrum ( max ¼ 338nm) compared to the apo form ( max ¼ 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 Ca2þ bond, the tryptophan lifetime can be described using two exponential decay model , with 1 ¼ 1.5 ns and 1 =3.5 ns. The Ca 2þ association to protein does not significantly alter tryptophan lifetime but increase the fraction of protein with the lifetime of 1.5 ns.
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