Characterization of the Photophysical, Thermodynamic and Structural Properties of the Terbium(III)-KChIP3 Complex

Abstract

KChIP3 (K+ channel interacting protein 3) is a calcium binding protein and active modulator of Kv4 channels in neuronal cells as well as a novel Ca2+ regulated transcriptional modulator. KChIP3(calsenilin) may also be involved in Alzheimer's disease through prevention of presenilin-2 fragmentation. Many of the KChIP3 interactions with its binding partners (Kv4, calmodulin, DNA, and drugs) have been shown to be dependent on calcium. Therefore, understanding the structural changes induced by Ca2+ is of utmost relevance to elucidating the mechanism of calcium signal transduction. Here, we show that the fluorescence emission and excitation spectra of the calcium luminescent analog Tb3+ is enhanced upon binding to the EF-hand of KChIP3, likely due to a mechanism of energy transfer between Phe/Trp and Tb3+. We also observe that unlike Tb3+ bound calmodulin, the luminescence lifetime of terbium bound to KChIP3 decays as a complex multiexponential (τaverage ∼ 1.8 ms) and is sensitive to the protein structure and drug (NS5806) binding. Using isothermal calorimetry we have determined that Tb3+ binds at least to four binding sites (Kd ∼ 2.2 μM) and is able to displace bound Ca2+ through an entropically driven mechanism (ΔH ∼ 3 kcal/mol). Secondary structural analysis of KChIP3 using far-UV CD spectroscopy shows that binding of Tb3+ induces the formation of an intermediate structure with less alpha helical content than that induced by Ca2+. However, using the hydrophobic probe 1,8-ANS we show that the structural changes induced by Tb3+ are large enough to expose a hydrophobic surface on KChIP3 identical to Ca2+ bound protein. Similarly to Ca2+, terbium binding also induces the dimerization of KChIP3. Overall, these results support the use of Tb3+ as an alternative fluorescent label in the study of Ca2+ induced structural changes in KChIP3.