Graduate Thesis Or Dissertation

 

Selenoprotein W : Purification and characterization of its interaction with calmodulin Public Deposited

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  • Selenoprotein W (SeW) is a protein whose function is unknown, but potentially plays a vital role in calcium metabolism, as an indirect link has been established with white muscle disease (WMD). White muscle disease occurs in selenium deficient animals, and is characterized by the precipitation of calcium in muscle, leading to paralysis and death. This thesis details efforts to purify and characterize SeW. This includes investigations into calcium binding, phosphorylation, and interaction with calmodulin (CaM). The main portion of the thesis consists of two manuscripts, the first dealing with the purification and production of SeW with and without bound glutathione, the second manuscript addresses SeW-CaM interaction. Supplemental material as well as the results of calcium binding studies and phosphorylation studies, are located in the appendices. Abstracts from the two manuscripts, follow. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) and electrospray ionization mass spectrometry (ESI MS) analysis of a 6x Histagged recombinant form of rat mutant selenoprotein W (RMSW) reveals that aerobic growth conditions primarily produce a form of RMSW without bound glutathione (10305 Da) whereas anaerobic conditions produce a glutathionebound (305 Da) form (10610 Da). Purification of RMSW was achieved with a procedure employing acetone precipitation and DEAE-cellulose chromatography, in addition to Ni-NTA agarose chromatography. Additional steps, including polyvalent metal ion binding (PMIB) resin chromatography and CM-cellulose chromatography, were necessary after elution from the Ni-NTA agarose column, in order to maintain solubility of the purified protein. Experiments applying partially purified extracts containing either rat mutant selenoprotein W (RMSW, Selenocysteine → cysteine, His₆ tag) or native rabbit selenoprotein W (SeW) to a calmodulin-sepharose column revealed that SeW interacts with calmodulin in a calcium dependent manner. Fluorescence polarization experiments with fluorescently labeled calmodulin and purified RMSW with and without bound glutathione revealed a Kd of 1.3 ± .1 X 10⁻⁶M for both forms of the protein. Competitive binding assays with myosin light chain kinase (MLCK) and fluorescently labeled calmodulin were performed for three peptide sequences (Nterm: GYKPKYLQLKEKL-NH2:, Rmid: VTVAGKLVHSKKRG-NH2, and Cterm: KFRKLVTAIKAALAQ-NH₂) which occur within proposed calmodulin binding sequences in rat SeW. The concentration of each peptide at which half-displacement of MLCK was achieved ([P]₅₀) was determined to be 5.5 nM, 6.3 nM, and 1.60 nM respectively. These values were used to estimate the dissociation constant of the peptide-calmodulin complex (Kr). The K values for Nterm and Rmid were determined to be < I nM, whereas the K for Cterm was determined to be 18 nM. In addition, during a preliminary test for specifity, a 100 fold excess of beef cardiac troponin C, a protein related to calmodulin, was unable to outcompete calmodulin for RMSW.
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