Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) is a Ser/Thr kinase necessary for long-term memory formation and other Ca 2+ -dependent signaling cascades such as fertilization. Here, we investigated the stability of CaMKIIα using a combination of differential scanning calorimetry (DSC), X-ray crystallography, and mass photometry (MP). The kinase domain has a low thermal stability (apparent T m = 36 C), which is slightly stabilized by ATP/MgCl 2 binding (apparent T m = 40 C) and significantly stabilized by regulatory segment binding (apparent T m = 60 C). We crystallized the kinase domain of CaMKII bound to p-coumaric acid in the active site. This structure reveals solvent-exposed hydrophobic residues in the substrate-binding pocket, which are normally buried in the autoinhibited structure when the regulatory segment is present. This likely accounts for the large stabilization that we observe in DSC measurements comparing the kinase alone with the kinase plus regulatory segment. The hub domain alone is extremely stable (apparent T m~9 0 C), and the holoenzyme structure has multiple unfolding transitions ranging from~60 C to 100 C. Using MP, we compared a CaMKIIα holoenzyme with different variable linker regions and determined that the dissociation of both these holoenzymes occurs at a higher concentration (is less stable) compared with the hub domain alone. We conclude that within the context of the holoenzyme structure, the kinase domain is stabilized, whereas the hub domain is destabilized. These data support a model where domains within the holoenzyme interact. K E Y W O R D SCaMKII, differential scanning calorimetry, mass photometry, oligomer dissociation, thermal stability
Spectrin repeat domains are a highly conserved biological motif found in many human structural proteins. Dystrophin contains 24 tandem spectrin repeats which provide structural support through mediating interactions between intracellular actin filaments and the extracellular matrix. However, the molecular mechanism by which dystrophin provides this support is unknown. Understanding this underlying structure/function relationship is important because mutations in dystrophin directly cause muscular dystrophy. Thus far, the following constructs have been expressed in E. coli, purified using chromatography, and thermodynamically characterized: S 17 , S 17-18, S 17-19. Parameters were determined through globally fitting thermal denaturation signals from Fluorescence Spectroscopy (FS), Circular Dichroism (CD), and Fluorescence Lifetime Spectroscopy (FLT) to a two-state model of unfolding. Fits were constrained using DC p values determined using Differential Scanning Calorimetry (DSC). This parameterization then allowed for determination of the free energy of stability (DG unfolding) of each construct. Results indicate that the DG unfolding of S 17 is nearly double that of S 17-19. This pronounced non-additivity indicates that tandem spectrin repeats mutually destabilize each other, termed negative coupling. Additionally, the comparison of Electron Paramagnetic Resonance (EPR) spectra of S 17 and S 17-19 indicate that the trimer exhibits greater local confirmational flexibility, consistent with decreased stability. To further test this negative coupling hypothesis, we are purifying S 19 for thermodynamic characterization. This will allow for the comparison of the sum of S 19 and S 17-18 's DG unfolding values with that of the trimer, helping further reveal the energetic and structural basis of dystrophin's mechanism.
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