Dissertation/Thesis Abstract

Functional Analysis of Calmodulin's Calcium Dependent Inactivation of Orai1
by Jensen, Drake, M.S., Southern Illinois University at Edwardsville, 2015, 99; 1589551
Abstract (Summary)

Calmodulin (CaM) plays an important role in calcium (Ca2+)-dependent signal transduction. Ca2+ binding to CaM triggers a conformational change, forming a hydrophobic patch that is important for target protein recognition. CaM regulates a Ca2+-dependent inactivation (CDI) process in store-operated Ca2+ entry (SOCE), by interacting with the N-terminus of the hexameric plasma membrane Ca2+ channel Orai1. To understand the relationship between Ca2+-induced hydrophobicity of CaM and the CaM/Orai interaction, chimera proteins constructed by exchanging EF-hands of CaM with those of Troponin C (TnC) were used as an informative probe to better understand the functionality of each EF-hand. ANS was used to assess the context of the induced hydrophobic surface on CaM and chimeras upon Ca2+ binding. The exchanged EF-hands from TnC to CaM resulted in reduced hydrophobicity compared with wild-type CaM, as depicted by ANS fluorescence and binding affinity. Such a conclusion is consistent with general concepts about the inadequacy of hydrophobic exposure for chimeras. However, such ANS responses exhibited no correlation with the ability to interact with Orai1. ANS lifetime measurements indicated that there are two types of ANS molecules with rather distinct fluorescence lifetimes, each specifically corresponding to one lobe of CaM or chimeras. Thermodynamic studies indicated the interaction between CaM and a 24-residue peptide corresponding to the CaM-binding domain of Orail1 (Orai-CMBD) is a 1:2 CaM/Orai-CMBD binding, in which each peptide binding yields a similar enthalpy change (ΔH = − 5.02 ± 0.13 kcal/mol) and binding affinity (Ka = 8.92 ± 1.03 x 105 M−1). With the exchanged EF1 and EF2, the resulting chimeras noted as CaM(1TnC) and CaM(2TnC), displayed a two sequential binding mode with a one-order weaker binding affinity and lower ?H than that of CaM, while CaM(3TnC) and CaM(4TnC) had similar binding thermodynamics as CaM. Circular Dichroism studies suggested differences in binding most likely resulted from changes in chimera three-dimensional structure rather than secondary structure, as the extent of ?-helical content from apo-, Ca2+-, and Orai-CMBD-bound proteins remained similar. The dissociation rate constant for CaM/Orai-CMBD was determined to be 1.41 ± 0.08 s−1 by rapid kinetics. Stern-Volmer plots of Orai-CMBD Trp76, indicated that the residue is located in a very hydrophobic environment but becomes more solvent accessible when EF1 and EF2 were exchanged. Here, the model of 1:2 binding stoichiometry of CaM/Orai-CMBD established in solution supports the unique, open binding mode suggested by already published structural studies.

Indexing (document details)
Advisor: Wei, Chin-Chuan
Commitee: Fowler, Tom, O'Brien, Leah
School: Southern Illinois University at Edwardsville
Department: Chemistry
School Location: United States -- Illinois
Source: MAI 54/05M(E), Masters Abstracts International
Source Type: DISSERTATION
Subjects: Biology, Biochemistry, Biophysics
Keywords: Calmodulin, Fluorescence, Isothermal titration calorimetry, Kinetics, Orai1, Store operated calcium entry
Publication Number: 1589551
ISBN: 978-1-321-77325-5
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