Skip to Main Content
 

Global Search Box

 
 
 

ETD Abstract Container

Abstract Header

Approach for Identification of Binding Proteins of Calcium Mobilizing Second Messengers: NAADP and cADPR

Abstract Details

2018, Doctor of Philosophy (PhD), University of Toledo, Medicinal Chemistry.
Nicotinic acid adenine dinucleotide phosphate (NAADP) and cyclic adenosine diphosphate-ribose (cADPR) are the two fraternal twins that act as Ca2+ mobilizing second messengers apart from inositol triphosphate (IP3). These Ca2+ mobilizing second messengers regulate various cellular functions like fertilization, insulin secretion, muscle contraction, etc. but the binding proteins for NAADP and cADPR are unknown. There is a high requisite to know about the binding proteins of NAADP and cADPR. To isolate and purify soluble mammalian NAADP binding proteins we propose to use ligand-affinity chromatography. Affinity chromatography is an isolation technique for proteins using `specific-ligand’ approach, by immobilizing an analog of NAADP to a hydrophilic water insoluble resin using a hydrophilic tetraethyleneglycol spacer arm. This affinity resin absorbs the specific proteins on to the resin which can be then eluted by higher ionic strength solvent or using natural ligand NAADP. Affinity chromatography will enable us to obtain the binding proteins of NAADP to identify the proteins by sequencing. To isolate and purify the cADPR binding proteins we propose to synthesize the bifunctional photoaffinity probes consisting of a photoaffinity label and purification site (clickable site). These photoaffinity probes should contain most of structural properties of naturally occurring substrate plus an added photoreactive group (aromatic azide) which has the potential to form an irreversible covalent bond with an amino acid residue located within the active site of the binding protein and an acetylene moiety to conjugate with an azide linked fluorescent or affinity tags for further purification of the protein. This affinity tag will then permit us to rapidly and efficiently enrich for the derivatized target protein. As a bifunctional cADPR analog we synthesized 8-N3-2'-O-propargyl-cADPR by alkylation of 8-bromoadenosine by propargyl bromide, followed by substitution of bromide with hydrazine by treating with anhydrous hydrazine and by diazotization hydrazine was converted to azide. The adenosine derivative was then phosphorylated and coupling to nicotinamide mononucleotide (NMN) to produce 8-N3-2'-O-propargyl-NAD+. The NAD+ analog was then cyclized enzymatically using Aplysia californica ADP ribosyl cyclase to produce the desire bifunctional 8-N3-2'-O-propargyl-cADPR analog.
James Slama (Committee Chair)
Zahoor Shah (Committee Member)
viranga Tillekeratne (Committee Member)
David Giovannucci (Committee Member)
158 p.

Recommended Citations

Citations

  • Andy, D. (2018). Approach for Identification of Binding Proteins of Calcium Mobilizing Second Messengers: NAADP and cADPR [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1525202591650673

    APA Style (7th edition)

  • Andy, Divya. Approach for Identification of Binding Proteins of Calcium Mobilizing Second Messengers: NAADP and cADPR. 2018. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1525202591650673.

    MLA Style (8th edition)

  • Andy, Divya. "Approach for Identification of Binding Proteins of Calcium Mobilizing Second Messengers: NAADP and cADPR." Doctoral dissertation, University of Toledo, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=mco1525202591650673

    Chicago Manual of Style (17th edition)