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Several short stories are presented including "Faeri Tayle," by Ling Ritter; " Artemis' 21st Birthday," by Madeline C. Shea and also presents a poem "I am a Coral Reef," by Carly Bowling.

Calmodulin is an EF‐hand calcium‐binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N‐ and C‐terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N‐domain fragment of ParameciumCaM (PCaM1–75), the addition of linker residues 76 to 80 (MKEQD) raised the Tmby 9°C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., Biochemistry 2002;41:15–20), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1–74to PCaM1–80). For the first 4, PCaM1–74through PCaM1–77, single amino acid additions at the C‐terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5°C in Tmand 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1–77through PCaM1–80were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N‐domain fragments. Comparisons of NMR (HSQC) spectra of 15N‐PCaM1–74and 15N‐PCaM1–80and analysis of high‐resolution structural models suggest these residues are “latched” to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains. Proteins 2003;50:381–391. © 2003 Wiley‐Liss, Inc.

Calmodulin is an EF-hand calcium-binding protein (148 a.a.) essential in intracellular signal transduction. Its homologous N- and C-terminal domains are separated by a linker that appears disordered in NMR studies. In a study of an N-domain fragment of Paramecium CaM (PCaM1–75), the addition of linker residues 76 to 80 (MKEQD) raised the Tm by 9°C and lowered calcium binding by 0.54 kcal/mol (Sorensen et al., Biochemistry 2002;41:15–20), showing that these tether residues affect energetics as well as being a barrier to diffusion. To determine the individual contributions of residues 74 through 80 (RKMKEQD) to stability and calcium affinity, we compared a nested series of 7 fragments (PCaM1–74 to PCaM1–80). For the first 4, PCaM1–74 through PCaM1–77, single amino acid additions at the C-terminus corresponded to stepwise increases in thermostability and decreases in calcium affinity with a net change of 13.5°C in Tm and 0.55 kcal/mol in free energy. The thermodynamic properties of fragments PCaM1–77 through PCaM1–80 were nearly identical. We concluded that the 3 basic residues in the sequence from 74 to 77 (RKMK) are critical to the increased stability and decreased calcium affinity of the longer N-domain fragments. Comparisons of NMR (HSQC) spectra of ¹⁵N-PCaM1–74 and ¹⁵N-PCaM1–80 and analysis of high-resolution structural models suggest these residues are “latched” to amino acids in helix A of CaM. The addition of residues E78, Q79, and D80 had a minimal effect on sites I and II, but they may contribute to the mechanism of energetic communication between the domains. Proteins 2003;50:381–391. © 2003 Wiley-Liss, Inc.

Ca(2+) influx through the N-methyl-d-aspartate (NMDA)-type glutamate receptor leads to activation and postsynaptic accumulation of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and ultimately to long term potentiation, which is thought to be the physiological correlate of learning and memory. The NMDA receptor also serves as a CaMKII docking site in dendritic spines with high affinity binding sites located on its NR1 and NR2B subunits. We demonstrate that high affinity binding of CaMKII to NR1 requires autophosphorylation of Thr(286). This autophosphorylation reduces the off rate to a level (t(12) = approximately 23 min) that is similar to that observed for dissociation of the T286D mutant CaMKII (t(12) = approximately 30 min) from spines after its glutamate-induced accumulation (Shen, K., Teruel, M. N., Connor, J. H., Shenolikar, S., and Meyer, T. (2000) Nat. Neurosci. 3, 881-886). CaMKII as well as the previously identified NR1 binding partners calmodulin and alpha-actinin bind to the short C-terminal portion of the C0 region of NR1. Like Ca(2+)/calmodulin, autophosphorylated CaMKII competes with alpha-actinin-2 for binding to NR1. We conclude that the NR1 C0 region is a key site for recruiting CaMKII to the postsynaptic site, where it may act in concert with calmodulin to modulate the stimulatory role of alpha-actinin interaction with the NMDA receptor.

Cooperative calcium binding to the two homologous domains of calmodulin (CaM) induces conformational changes that regulate its association with and activation of numerous cellular target proteins. Calcium binding to the pair of high-affinity sites (III and IV in the C-domain) can be monitored by observing calcium-dependent changes in intrinsic tyrosine fluorescence intensity (λex/λem of 277/320nm). However, calcium binding to the low-affinity sites (I and II in the N-domain) is more difficult to measure with optical spectroscopy because that domain of CaM does not contain tryptophan or tyrosine. We recently demonstrated that calcium-dependent changes in intrinsic phenylalanine fluorescence (λex/λem of 250/280nm) of an N-domain fragment of CaM reflect occupancy of sites I and II (VanScyoc, W. S., and M. A. Shea, 2001, Protein Sci. 10:1758–1768). Using steady-state and time-resolved fluorescence methods, we now show that these excitation and emission wavelength pairs for phenylalanine and tyrosine fluorescence can be used to monitor equilibrium calcium titrations of the individual domains in full-length CaM. Calcium-dependent changes in phenylalanine fluorescence specifically indicate ion occupancy of sites I and II in the N-domain because phenylalanine residues in the C-domain are nonemissive. Tyrosine emission from the C-domain does not interfere with phenylalanine fluorescence signals from the N-domain. This is the first demonstration that intrinsic fluorescence may be used to monitor calcium binding to each domain of CaM. In this way, we also evaluated how mutations of two residues (Arg74 and Arg90) located between sites II and III can alter the calcium-binding properties of each of the domains. The mutation R74A caused an increase in the calcium affinity of sites I and II in the N-domain. The mutation R90A caused an increase in calcium affinity of sites III and IV in the C-domain whereas R90G caused an increase in calcium affinity of sites in both domains. This approach holds promise for exploring the linked energetics of calcium binding and target recognition.

Calmodulin activates the skeletal muscle Ca(2+) release channel RYR1 at nm Ca(2+) concentrations and inhibits the channel at microm Ca(2+) concentrations. Using a deletion mutant of calmodulin, we demonstrate that amino acids 2-8 are required for high affinity binding of calmodulin to RYR1 at both nm and microm Ca(2+) concentrations and are required for maximum inhibition of the channel at microm Ca(2+) concentrations. In contrast, the addition of three amino acids to the N terminus of calmodulin increased the affinity for RYR1 at both nm and microm Ca(2+) concentrations, but destroyed its functional effects on RYR1 at nm Ca(2+). Using both full-length RYR1 and synthetic peptides, we demonstrate that the calmodulin-binding site on RYR1 is likely to be noncontiguous, with the C-terminal lobe of both apocalmodulin and Ca(2+)-calmodulin binding to amino acids between positions 3614 and 3643 and the N-terminal lobe binding at sites that are not proximal in the primary sequence. Ca(2+) binding to the C-terminal lobe of calmodulin converted it from an activator to an inhibitor, but an interaction with the N-terminal lobe was required for a maximum effect on RYR1. This interaction apparently depends on the native sequence or structure of the first few amino acids at the N terminus of calmodulin.

Oxidant stress can contribute to health and disease. Here we show that invertebrates and vertebrates share a common stereospecific redox pathway that protects against pathological responses to stress, at the cost of reduced physiological performance, by constraining CaMKII activity. MICAL1, a methionine oxygenase thought to exclusively target actin, and MSRB, a methionine reductase, control the stereospecific redox status of M308, a highly conserved residue in the calmodulin (CaM) binding domain of CaMKII. Oxidized or mutant M308 (M308V) decreased CaM binding and CaMKII activity, while absence of MICAL1in mice caused heart failure and premature death due to CaMKII hyperactivation. Mimicking the effects of M308 oxidation decreased fight or flight responses in mice, strikingly impaired heart function in Drosophila melanogasterand caused disease protection in human induced pluripotent stem cell-derived cardiomyocytes with catecholaminergic polymorphic ventricular tachycardia, a CaMKII-sensitive genetic arrhythmia syndrome. Our studies identify a new stereospecific redox pathway that regulates cardiac physiological and pathological responses to stress across species.

Calcium and lipids stimulate the kinase Itk through the same domain to promote proinflammatory T cell signaling.