Date of Award

Summer 6-27-2013

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

Abstract

Calmodulin (CaM) is a eukaryotic Ca2+ signaling protein which can interact with more than 300 enzymes in the cell including membrane proteins Ryanodine receptor1 (RyR1) and gap junction protein connexin 43 (Cx43). By binding to Ca2+, CaM undergoes a conformational change which exposed the hydrophobic patch that access to the target protein. wt-CaM and three mutant CaM (isolate C and N domain of CaM, deletion of five residues from the central linker of CaM) are designed for studying the specific contributions to calcium binding affinity and calcium induced conformational change. wt-CaM exhibits metal binding affinity to calcium analog Tb3+ with a Kd of 3.97 nM using FRET assay and metal-buffer system and activates target protein phosphodiesterase assay. The Kd values of domain specific calcium binding affinity of CaM probed by intrinsic Phe or Tyr are 12.2 and 2.77 uM, respectively. In addition, Ca2+ also induces helicity for both w.t. CaM and C-terminal domain variant. Further, conditions such as medium and Glucose amount for isotopic labeling of CaM by 15N, 13C and D2O have been optimized with relatively high yield of hetero-isotopic labeled CaM. This prepared us to probe the detailed interaction of CaM and its target protein and calcium induced conformational change by high resolution NMR. Furthermore, RyR1 mini domain, which contains two CaM binding regions of RyR1 was designed to study the binding mode of the two regions with CaM. Obtaining bacterial expressed and purified RyR1 mini domain was achieved by engineered with a His-Tag which overcomes the insoluble issue that occurred in the initial study of expression and purification with a GST tag. Moreover, to probe the interaction of CaM to the cytosolic loop of Cx43 that contains two putative CaM binding sites as well as the role of transmembrane region of Cx43, we have successfully expressed and purified fragments Cx4388-154 and Cx4399-154 as a His-tag protein encompassing regions 88-154 and 99-154 of Cx43 with predicted CaM binding sites with and without additional transmembrane region from Cx43. Both fragments were obtained with high yield after expressed as inclusion body and His-tag purification. Fragment Cx4388-154 was shown to bind dansylated CaM with a Kd of 0.107 μM using florescence spectroscopy.

DOI

https://doi.org/10.57709/4351398

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