Dissertation/Thesis Abstract

Characterization of Interaction of Calmodulin and its Chimeras with Orai1
by Shakya, Shubha, M.S., Southern Illinois University at Edwardsville, 2013, 65; 1544484
Abstract (Summary)

Calcium Dependent Inactivation (CDI) is a key regulator of calcium release-activated calcium channels. The channel subunit Orai1 is demonstrated to interact with calmodulin (CaM) and evoke CDI. The CaM-binding sequence of Orai1 (Orai1 pep) is identified, but the binding mechanism is not clear. Here, we constructed CaM and its four Troponin C chimeras (TnCs). TnCs were created by substituting the Ca2+ binding EF hand motif of CaM with the corresponding EF hand of TnC. We characterized the Ca2+ binding properties of CaM and its Chimeras with the help of fluorescence and stopped flow kinetics. The chimeras retained their property to expose a hydrophobic patch upon Ca 2+ binding and the Ca2+ dissociation rates were also found to be similar. The interaction of CaM/chimeras with Orai1 pep was characterized by fluorescence spectroscopy, which indicated a significant hydrophobic increase upon Orai1 pep binding. Such an interaction is Ca2+ dependent. Then, we studied interactions between the CaM/chimeras and Orai1 pep using Isothermal Titration Calorimetry (ITC). ITC revealed that the binding of CaM to Orai1 pep is moderate with Ka= 9.5 × 10 5 M-1. The binding constant for 1TnC and 2TnC is approximately one order lower due to the decrease in binding entropy. These data, combined with the results from quenching studies, suggested that the W76 residue of Orai1 pep plays an important role in binding and the negative charges on the C-terminal of CaM are not critical for peptide binding. However, more hydrogen bonds and van der Waal forces are induced in 3TnC and 4TnC by peptide binding.

Indexing (document details)
Advisor: Wei, Chin-Chuan
Commitee: Lu, Yun, O'Brien, Leah
School: Southern Illinois University at Edwardsville
Department: Chemistry
School Location: United States -- Illinois
Source: MAI 52/02M(E), Masters Abstracts International
Subjects: Biochemistry
Keywords: Calcium release activated calcium channel, Calmodulin, Calmodulin chimeras, Store operated channels, Troponin c
Publication Number: 1544484
ISBN: 978-1-303-34761-0
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