Characterization of the Photophysical, Thermodynamic, and Structural Properties of the Terbium(III)–DREAM Complex

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• 作者：Walter G. Gonzalez ; Victoria Ramos ; Maurizio Diaz ; Alyssa Garabedian ; Juan Camilo Molano-Arevalo ; Francisco Fernandez-Lima ; Jaroslava Miksovska
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：March 29, 2016
• 年：2016
• 卷：55
• 期：12
• 页码：1873-1886
• 全文大小：730K
• ISSN：1520-4995

文摘

DREAM (also known as K⁺ channel interacting protein 3 and calsenilin) is a calcium binding protein and an active modulator of K_V4 channels in neuronal cells as well as a novel Ca²⁺-regulated transcriptional modulator. DREAM has also been associated with the regulation of Alzheimer’s disease through the prevention of presenilin-2 fragmentation. Many interactions of DREAM with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by binding of metals to DREAM is essential for elucidating the mechanism of signal transduction and biological activity of this protein. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analogue, Tb³⁺, are enhanced upon binding to the EF-hands of DREAM due to a mechanism of energy transfer between Trp and Tb³⁺. We also observe that unlike Tb³⁺-bound calmodulin, the luminescence lifetime of terbium bound to DREAM decays as a complex multiexponential (τ_average ∼ 1.8 ms) that is sensitive to perturbation of the protein structure and drug (NS5806) binding. Using isothermal calorimetry, we have determined that Tb³⁺ binds to at least three sites with high affinity (K_d = 1.8 μM in the presence of Ca²⁺) and displaces bound Ca²⁺ through an entropically driven mechanism (ΔH ∼ 12 kcal mol^–1, and TΔS ∼ 22 kcal mol^–1). Furthermore, the hydrophobic probe 1,8-ANS shows that Tb³⁺, like Ca²⁺, triggers the exposure of a hydrophobic surface on DREAM, which modulates ligand binding. Analogous to Ca²⁺ binding, Tb³⁺ binding also induces the dimerization of DREAM. Secondary structural analyses using far-UV circular dichroism and trapped ion mobility spectrometry–mass spectrometry reveal that replacement of Ca²⁺ with Tb³⁺ preserves the folding state with minimal changes to the overall structure of DREAM. These findings pave the way for further investigation of the metal binding properties of DREAM using lanthanides as well as the study of DREAM–protein complexes by lanthanide resonance energy transfer or nuclear magnetic resonance.