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.
|Commitee:||Lu, Yun, O'Brien, Leah|
|School:||Southern Illinois University at Edwardsville|
|School Location:||United States -- Illinois|
|Source:||MAI 52/02M(E), Masters Abstracts International|
|Keywords:||Calcium release activated calcium channel, Calmodulin, Calmodulin chimeras, Store operated channels, Troponin c|
Copyright in each Dissertation and Thesis is retained by the author. All Rights Reserved
The supplemental file or files you are about to download were provided to ProQuest by the author as part of a
dissertation or thesis. The supplemental files are provided "AS IS" without warranty. ProQuest is not responsible for the
content, format or impact on the supplemental file(s) on our system. in some cases, the file type may be unknown or
may be a .exe file. We recommend caution as you open such files.
Copyright of the original materials contained in the supplemental file is retained by the author and your access to the
supplemental files is subject to the ProQuest Terms and Conditions of use.
Depending on the size of the file(s) you are downloading, the system may take some time to download them. Please be