Poster abstracts

Poster number 17 submitted by Shane Walton

Engineering a therapeutic calmodulin: Cation binding and target enzyme interaction in soybean and Arabidopsis

Shane D. Walton (Physiology and Cell Biology, The Ohio State University), Vikram Shettigar (Physiology and Cell Biology, The Ohio State University), Harshini Chakravarthy (Physiology and Cell Biology, The Ohio State University), Andrew J. ONeil (Physiology and Cell Biology, The Ohio State University), Cory W. Wilson (Physiology and Cell Biology, The Ohio State University), Jonathan P. Davis (Physiology and Cell Biology, The Ohio State University)

Abstract:
Calmodulin (CaM) is a ubiquitous decoder of the calcium signal within the animal and plant kingdoms. All vertebrates encode a single isoform of CaM, while plants encode up to ten different isoforms, each of which differs in their binding, activation or inhibition of specific target enzymes in response to stimuli. Examples of these stimuli include pathogen attack and wounding, as well as chemical, thermal, and ionic stresses. Our goal is to adapt the design principles that the plant has evolved to selectively modulate the CaM-dependent enzyme hubs within the mammalian cell.

As a first step in understanding the functional differences among these CaMs, we have studied the Ca²⁺ binding properties of two unique soybean CaMs: sCaM1 and sCaM4, as well as three unique Arabidopsis thaliana CaMs: CaM4, CaM5 and CaM7. We performed steady state fluorescence experiments to follow cation binding and used the stopped-flow technique to measure the kinetics of cation dissociation from these CaMs by means of a variety of fluorescent probes.

We have previously demonstrated that two soybean CaMs bound to several important mammalian enzymes, some of which were inhibited instead of activated by CaM binding. In this study we performed stopped-flow experiments to determine the effect of target binding on the Ca²⁺ dissociation rates for these various CaMs, as target binding changes CaM’s affinity for Ca²⁺, drastically slowing the Ca²⁺ dissociation rate.

For each CaM, our analysis revealed that the rate of Ca²⁺ dissociation was similar to the regional structural change occurring at each domain, which in turn was similar to the rate of closure of each domain’s hydrophobic pocket. All soybean and Arabidopsis CaMs displayed distinct binding behavior for a variety of mammalian target enzymes. These studies can pave the way for further use of these CaMs for potential applications involving activation or inhibition of a subset of CaM-regulated enzymes.

This work was supported by AHA SDG (JPD)

Keywords: Calcium-binding, Calmodulin, Protein engineering