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Calcium and EDTA Induced Folding and Unfolding of Calmodulin on Functionalized Quantum Dot Surfaces

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Abstract:

Calcium is an ubiquitous second messenger signal that is critical to many cellular processes. As such considerable efforts have been made to develop sensitive high resolution calcium sensors. However, organic dye calcium sensors have inherent limitations in signal to noise ratio and spatial resolution. We have developed a novel quantum dot (qdot) based calcium sensor with superior optical properties for biological detection of functional calcium signaling. Here, we discuss the in vitro calcium binding properties of our sensor. The sensor was designed as a fluorescence resonance energy transfer (FRET) complex, composed of a dihydrolipoic acid capped quantum (DHLA-qdot) which acts as a fluorescence donor, an organic dye (Alexa Fluor 647) which acts as a fluorescence acceptor, and calmodulin (CaM) which serves as the active calcium sensing element. We confirmed that a significant FRET signal was observed between the donor (610 nm) and acceptor (670 nm) emission wavelengths upon binding with calcium ions with a maximal fractional change in FRET ratio (ΔR/R) of up to 6.6. Unlike this DHLA-qdot system, almost no FRET was observed when qdots were coated with the relatively large surface ligands polyethylene glycol and polyethyleneimine, suggesting negative effects of the bulky ligands on CaM folding. Calcium-induced FRET in our system was reversed by EDTA treatment, suggesting the unfolding of CaM on the nanoparticle surface in the absence of calcium, enabling a regeneration of the sensor system.

Keywords: CALCIUM; CALCIUM SENSOR; CALMODULIN; EDTA; FOLDING AND UNFOLDING; FRET; QUANTUM DOT

Document Type: Research Article

DOI: https://doi.org/10.1166/jns.2012.1017

Publication date: 2012-04-01

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