Subunit Exchange in CaMKII Alpha Variant

Presenter

Laura Ann Jansen

Campus

UMass Amherst

Sponsor

Margaret Stratton, Department of Biochemistry and Molecular Biology, UMass Amherst

Schedule

Session 3, 1:30 PM - 2:15 PM [Schedule by Time][Poster Grid for Time/Location]

Location

Poster Board A45, Campus Center Auditorium, Row 3 (A41-A60) [Poster Location Map]

Abstract

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) holoenzyme is a Ser/Thr multimeric kinase made up of symmetric rings stacked on one another. The most common is two hexamers stacked to make a dodecamer. Extending out from the hub is the linker region and regulatory segment, which attaches to the kinase domain. It is known that CaMKII is required for long-term memory potentiation (Stratton, 2014). However, the allosteric communication mechanism of the CaMKII Alpha structure is still unknown, and mutated variants of this structure will aid in learning more about activation and subunit exchange.  

To further interrogate subunit exchange, a variant of CaMKII was designed to enable cross-linking of vertical hub dimers. This CaMKII Alpha variant was expressed in E. coli and purified. Activation and subunit exchange experiments were performed and the data was visualized by running agarose gels, SDS Page gels, and Mass Photometry to measure the molecular masses of single particles. When treated with DTNB, it was found that there was successful crosslinking of the dimer subunits, giving a holoenzyme. However, activation of the populations gave monomeric species, which was unexpected. Spontaneous, random cleavage of His Sumo tag resulted in the need for a different plasmid to be used in these experiments. Discovering more about subunit exchange and activation allows for a better understanding of the mechanisms of long-term memory potentiation.

Keywords

Subunit Exchange, CaMKII, Biochemistry, Memory

Research Area

Biological Organisms

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