Master of Science (M.Sc.)
Shantá D Hinton
The pseudophosphatase MK-STYX [MAPK (mitogen-activated protein kinase) -phosphoserine/threonine/tyrosine-interacting protein] is an atypical MKP (MAPK phosphatase). The DUSP (dual-specificity phosphatase) domain of MK-STYX lacks critical histidine and cysteine residues in the active site motif (HCX5R), rendering it catalytically inactive. Also important to the function of MKPs is the CH2 domain (cell division cycle 25 phosphatase homology 2 domain), which is interrupted by a KIM (kinase-interacting motif). Unlike the KIM of its MKP active homologs, MK-STYX lacks consecutive arginines for binding MKP target proteins. Despite this, MK-STYX has been shown to be a regulator of multiple pathways, including stress response, apoptosis, and neurite formation, and has been implicated in various cancers. Uncovering the macromolecular structure of MK-STYX is the key to understanding the function of the atypical domains of MK-STYX, what role MK-STYX plays in signaling pathways, why it induces particular phenotypes, and how this differs from other MKPs. Determining the structure of MK-STYX requires a combined approach of protein crystallography and bioinformatics. An investigation using computational approaches revealed that when MK-STYX is mutated to restore consecutive arginines in the KIM of MK-STYX back to that of active its active MKP homologs, it results in a different predicted binding pocket compared to the wild-type structure, supporting the idea that MK-STYX has a unique function. To obtain the X-ray crystallography structure of MK-STYX, sample preparation must be optimized before a crystal screen can commence. Multiple methods of purification using immobilized-metal affinity chromatography (IMAC) were tested, such as: fast protein liquid chromatography (FPLC), gravity-flow purification, spin-column purification, and batch purification. The elution fractions from these purifications were analyzed to reveal the one best suited to MK-STYX. The best purification of MK-STYX was achieved using the batch method of purification using cobalt resin, but two dominant non-specific proteins continued to co-purify with MK-STYX. These proteins were identified as 60kDa chaperonin (Cpn60) and the transcription termination factor Rho (Rho factor). Immunoprecipitation (IP) confirmed that these proteins were interacting with MK-STYX. Attempts at removing Cpn60 and Rho factor were made by supplementing the buffers used during purification with CHAPS detergent (3-((3-cholamidopropyl) dimethylammonio)-1-propanesulfonate), ATP (adenosine 5′-triphosphate), and urea. Using 8M urea to purify MK-STYX under denaturing conditions was the only variation that improved the purity of the elution fractions.
© The Author
Hepworth, Emma Marie Wilber, "Understanding The Function Of A MAPK-"STYX"-Domain Protein Tyrosine Phosphatase" (2022). Dissertations, Theses, and Masters Projects. William & Mary. Paper 1673281751.
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