Cluster analysis application identifies muscle characteristics of importance for beef tenderness

Background

Unsaturated fatty acids are susceptible to oxidation and damaged chains are removed from glycerophospholipids by phospholipase A₂. De-acylated lipids are then re-acylated by lysophospholipid acyltransferase enzymes such as LPCAT1 which catalyses the formation of phosphatidylcholine (PC) from lysoPC and long-chain acyl-CoA.

Results

Activity of LPCAT1 is inhibited by Ca²⁺, and a Ca²⁺-binding motif of the EF-hand type, EFh-1, was identified in the carboxyl-terminal domain of the protein. The residues Asp-392 and Glu-403 define the loop of the hairpin structure formed by EFh-1. Substitution of D³⁹² and E⁴⁰³ to alanine rendered an enzyme insensitive to Ca²⁺, which established that Ca²⁺ binding to that region negatively regulates the activity of the acyltransferase amino-terminal domain. Residue Cys-211 of the conserved motif III is not essential for catalysis and not sufficient for sensitivity to treatment by sulfhydryl-modifier agents. Among the several active cysteine-substitution mutants of LPCAT1 generated, we identified one to be resistant to treatment by sulfhydryl-alkylating and sulfhydryl-oxidizer agents.

Conclusion

Mutant forms of LPCAT1 that are not inhibited by Ca²⁺ and sulfhydryl-alkylating and ?Coxidizing agents will provide a better understanding of the physiological function of a mechanism that places the formation of PC, and the disposal of the bioactive species lysoPC, under the control of the redox status and Ca²⁺ concentration of the cell.     

Ca<Superscript>2+</Superscript> binding to complement-type repeat domains 5 and 6 from the low-density lipoprotein receptor-related protein

Background

The binding of ligands to clusters of complement-type repeat (CR)-domains in proteins of the low-density lipoprotein receptor (LDLR) family is dependent on Ca²⁺ ions. One reason for this cation requirement was identified from the crystal structure data for a CR-domain from the prototypic LDLR, which showed the burial of a Ca²⁺ ion as a necessity for correct folding and stabilization of this protein module. Additional Ca²⁺ binding data to other CR-domains from both LDLR and the LDLR-related protein (LRP) have suggested the presence of a conserved Ca²⁺ cage within CR-domains from this family of receptors that function in endocytosis and signalling.

Results

We have previously described the binding of several ligands to a fragment comprising the fifth and the sixth CR-domain (CR56) from LRP, as well as qualitatively described the binding of Ca²⁺ ions to this CR-domain pair. In the present study we have applied the rate dialysis method to measure the affinity for Ca²⁺, and show that CR56 binds 2 Ca²⁺ ions with an average affinity of K_D = 10.6 microM, and there is no indication of additional Ca²⁺ binding sites within this receptor fragment.

Conclusions

Both CR-domains of CR56 bind a single Ca²⁺ ion with an affinity of 10.6 microM within the range of affinities demonstrated for several other CR-domains.

     

Analysis of intercellular communication by flexible hydrodynamic gating on a microfluidic chip

Intercellular Ca²⁺ waves are propagation of Ca²⁺ transients among cells that could be initiated by chemical stimulation. Current methods for analyzing intercellular Ca²⁺ waves are difficult to realize localized chemical stimulations upon the target cell without interfering with adjacent contacting cells. In this paper, a simple and flexible microfluidic method was developed for investigating the intercellular communication of Ca²⁺ signals. A cross-patterned microfluidic chip was designed and fabricated with polydimethylsiloxane as the structural material. Localized chemical stimulation was achieved by a new strategy based on hydrodynamic gating technique. Clusters of target cells were seeded at the location within 300 μm downstream of the intersection of the cross-shaped microchannel. Confined lateral molecular diffusion largely minimized the interference from diffusion-induced stimulation of adjacent cells. Localized stimulation of the target cell with adenosine 5′-triphosphate successfully induced the propagation of intercellular Ca²⁺ waves among a population of adjacent contacting cells. Further inhibition studies verified that the propagation of calcium signals among NIH-3 T3 cells was dependent on direct cytosolic transfer via gap junctions. The developed microfluidic method provides a versatile platform for investigating the dynamics of intercellular communications.

Characterization of hydromedusan Ca2+-regulated photoproteins as a tool for measurement of Ca2+concentration

Calcium ion is a ubiquitous intracellular messenger, performing this function in many eukaryotic cells. To understand calcium regulation mechanisms and how disturbances of these mechanisms are associated with disease states, it is necessary to measure calcium inside cells. Ca²⁺-regulated photoproteins have been successfully used for this purpose for many years. Here we report the results of comparative studies on the properties of recombinant aequorin from Aequorea victoria, recombinant obelins from Obelia geniculata and Obelia longissima, recombinant mitrocomin from Mitrocoma cellularia, and recombinant clytin from Clytia gregaria as intracellular calcium indicators in a set of identical in vitro and in vivo experiments. Although photoproteins reveal a high degree of identity of amino acid sequences and spatial structures, and, apparently, have a common mechanism for the bioluminescence reaction, they were found to differ in the Ca²⁺ concentration detection limit, the sensitivity of bioluminescence to Mg²⁺, and the rates of the rise of the luminescence signal with a sudden change of Ca²⁺ concentration. In addition, the bioluminescence activities of Chinese hamster ovary cells expressing wild-type photoproteins also differed. The light signals of cells expressing mitrocomin, for example, slightly exceeded the background, suggesting that mitrocomin may be hardly used to detect intracellular Ca²⁺ without modifications improving its properties. On the basis of experiments on the activation of endogenous P2Y₂ receptor in Chinese hamster ovary cells by ATP, we suggest that wild-type aequorin and obelin from O. longissima are more suitable for calcium detection in cytoplasm, whereas clytin and obelin from O. geniculata can be used for calcium measurement in cell compartments with high Ca²⁺ concentration. Figure

Hydromedusan photoproteins differ in Ca²⁺ concentration detection limit, sensitivity of bioluminescence to Mg²⁺, and rates of rise of luminescence signal with a sudden change of [Ca²⁺] despite a high degree of identity of their amino acid sequences and spatial structures, and, apparently, a common mechanism for the bioluminescence reaction.     

Experimental and theoretical study on the complexation of Ca2+ with beauvericin

From extraction experiments and γ-activity measurements, the exchange extraction constant corresponding to the equilibrium Ca²⁺(aq) + 1·Sr²⁺(nb) ? 1·Ca²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (1 = beauvericin; aq = aqueous phase, nb = nitrobenzene phase) was evaluated as log K _ex(Ca²⁺, 1·Sr²⁺) = 1.1 ± 0.1. Further, the stability constant of the 1·Ca²⁺ complex in nitrobenzene saturated with water was calculated for a temperature of 25 °C: log β _nb(1·Ca²⁺) = 10.1 ± 0.2. Finally, by using quantum mechanical density functional level of theory calculations, the most probable structures of the non-hydrated 1·Ca²⁺ and hydrated 1·Ca²⁺·H₂O complex species were predicted.     

Heat capacity and density of solutions of calcium and cadmium nitrates in N-methylpyrrolidone at 298.15 K

The heat capacity and density of solutions of calcium and cadmium nitrates in N-methylpyrrolidone (MP) at 298.15 K are studied by calorimetry and densimetry. The obtained data are discussed in relation to certain features of solvation and complex formation in solutions of these salts. The standard partial molar heat capacities and volumes ( $\overline {C_{p^2 }^0 }$ and $\overline {V_2^0 }$ ) of the electrolytes in MP are calculated. The standard heat capacities $\overline {C_{p^i }^0 }$ and volumes $\overline {V_i^0 }$ of Ca²⁺ and Cd²⁺ ions in MP at 298.15 K were determined, along with the contribution from specific interactions to the values of $\overline {C_{p^i }^0 }$ and $\overline {V_i^0 }$ of Cd²⁺ ions in MP solution.     

New garnet compounds A 3 2+ B 2 2+ C4+V 2 5+ O12(A = Ca,Cd; B = Mg,Zn, Co,Ni, Cu,Mn, Cd; C = Ge,Si)

Garnet compounds A ₃ ²⁺ B ₂ ²⁺ C⁴⁺V ₂ ⁵⁺ O₁₂ (A = Ca, Cd; B = Mg, Zn, Co, Ni, Cu, Mn, Cd; C = Ge, Si) (space group \(Ia\bar 3d\) , Z = 8) have been prepared by solid-phase synthesis in air at 900–1250°C. Most of these compounds melt incongruently or decompose in the solid phase. The isomorphic capacity of garnets and their homogeneity fields are discussed. The structures of Ca₃Zn₂GeV₂O₁₂ and Ca₃Cu₂GeV₂O₁₂ have been refined by the Rietveld method.     

Structural Analysis of Guanylyl Cyclase-Activating Protein-2 (GCAP-2) Homodimer by Stable Isotope-Labeling, Chemical Cross-Linking, and Mass Spectrometry

The topology of the GCAP-2 homodimer was investigated by chemical cross-linking and high resolution mass spectrometry. Complementary conducted size-exclusion chromatography and analytical ultracentrifugation studies indicated that GCAP-2 forms a homodimer both in the absence and in the presence of Ca²⁺. In-depth MS and MS/MS analysis of the cross-linked products was aided by ¹⁵ ? N-labeled GCAP-2. The use of isotope-labeled protein delivered reliable structural information on the GCAP-2 homodimer, enabling an unambiguous discrimination between cross-links within one monomer (intramolecular) or between two subunits (intermolecular). The limited number of cross-links obtained in the Ca²⁺-bound state allowed us to deduce a defined homodimeric GCAP-2 structure by a docking and molecular dynamics approach. In the Ca²⁺-free state, GCAP-2 is more flexible as indicated by the higher number of cross-links. We consider stable isotope-labeling to be indispensable for deriving reliable structural information from chemical cross-linking data of multi-subunit protein assemblies.

Synthesis, 1H-NMR conformational analysis and complexation studies of two di-imidazolyl acetamido p-tert-butylcalix[4]arenes

The synthesis of a new di-imidazolyl-di-methoxy acetamido p-tert-butylcalix[4]arene 4 is reported. 4 has been prepared by reacting the corresponding di-methyl ester di-methoxy derivative with histamine in 1:1 mixture of methanol: toluene. The binding properties of 4 towards alkali, alkaline earth, transition (Zn²⁺, Co²⁺) and heavy (Pb²⁺, Cd²⁺) metals have been investigated along with the complexes stoichiometries. The ¹H-NMR spectra of complexes show the location of cations in receptor 4. Partial cone conformation is observed only with strontium and calcium whereas the cone conformation is detected with most of the cations. Comparison of the complexation results with those obtained for di-imidazolyl acetamido p-tert-butylcalix[4]arene 3 missing the methyl groups is also reported.     

Screening interaction between ochratoxin A and aptamers by fluorescence anisotropy approach

By taking advantage of the intrinsic fluorescence of ochratoxin A (OTA), we present a fluorescence anisotropy approach for rapid analysis of the interactions between OTA and aptamers. The specific binding of OTA with a 36-mer aptamer can induce increased fluorescence anisotropy (FA) of OTA as the result of the freedom restriction of OTA and the increase of molecular volume, and the maximum FA change is about 0.160. This FA approach enables an easy way to investigate the effects of buffer compositions like metal ions on the affinity binding. FA analysis shows the interaction between OTA and aptamer is greatly enhanced by the simultaneous presence of Ca²⁺ and Na⁺, while the binding affinity of aptamer decreases more than 18-fold when only Ca²⁺ exists, and the binding is completely lost when Ca²⁺ is absent. Crucial region of the aptamer for binding can be mapped through FA analysis and aptamer mutation. The demonstrated FA approach maintains the advantages of FA in simplicity, rapidity, and robustness. This investigation will help the development of aptamer-based assays for OTA detection in optimizing the binding conditions, modification of aptamers, and rational design.

Synergistic extraction of some divalent metal cations into nitrobenzene by using strontium dicarbollylcobaltate and electroneutral macrocyclic lactam receptor

From extraction experiments and $ \gamma $ -activity measurements, the exchange extraction constants corresponding to the general equilibrium M²⁺(aq) + 1·Sr²⁺(nb) $ \Leftrightarrow $ 1·M²⁺(nb) + Sr²⁺(aq) taking place in the two-phase water–nitrobenzene system (M²⁺ = Mg²⁺, Ca²⁺, Ba²⁺, Pb²⁺, Cu²⁺, Zn²⁺, Cd²⁺, $ {\hbox{UO}}_{2}^{2 + } $ , Mn²⁺, Co²⁺, Ni²⁺; 1 = macrocyclic lactam receptor–see Scheme 1; aq = aqueous phase, nb = nitrobenzene phase) were evaluated. Moreover, the stability constants of the 1·M²⁺ complexes in nitrobenzene saturated with water were calculated; they were found to increase in the following cation order: Mg²⁺ < Co²⁺ < Cu²⁺, Mn²⁺, Ni²⁺ < Cd²⁺ < Ca²⁺ < Ba²⁺, Zn²⁺ < Pb²⁺ <  $ {\hbox{UO}}_{2}^{2 + } $ .

Synthesis and Regioselective Hydrolysis of Novel Dialkyl 4-Imidazolyl-1,4-Dihydropyridine-3,5-dicaroxlates as Potential Dual Acting Angiotensin II Inhibitors and Calcium Channel Blockers

Most of the known effects of angiotensin II are mediated via AT₁ receptor by increasing intracellular Ca²⁺ by influx of extracellular Ca²⁺. Combination therapies of angiotensin receptor blocker (ARB) with calcium channel blocker (CCB) which act through L-type calcium channel have beneficial therapeutic and protective effects on cardiovascular system. Thus, it was hypothesized that merging the key structural elements present in an AT₁ receptor antagonist (telmisartan) with key structural elements in 1,4-dihydropyridine calcium channel blockers (nifedipine) would yield a compound with dual activity for both receptors. This strategy led to the design and synthesis of dialkyl 1,4-dihydro-2,6-dimethyl-4-[2-n-alkyl-1-[(2′-carboxybiphenyl- 4-yl)methyl]imidazole-4(or 5)-yl]-3,5-pyridinedicarboxylates (4 and 6). The synthesis of compounds 4 and 6 was accomplished through the reaction of 2-n-alkyl-1-[(2′-carbomethoxybiphenyl-4-yl)methyl]imidazole-4(or 5)-carboxaldehydes with alkyl acetoacetate followed by regioselctive hydrolysis of carboethoxybiopheny to carboxybiphenyl that are essential for ARB activity. It is suggested that existence of hindrance by substituted groups prevent hydrolysis of esteric groups on dihydropyridine ring. The structures of the compounds were characterized by ¹H-nuclear magnetic resonance, infrared and mass spectroscopy.     

Syntheses, spectroscopic characterization and metal ion binding properties of benzo-15-crown-5 derivatives and their sodium and nickel(II) complexes

New benzo-15-crown-5 derivatives containing nitro, amine and imine groups were prepared. Nitro compound (1) was prepared after the reaction?4′,5′-bis(bromethyl)benzo-15-crown-5 and o-nitrophenol in the presence of NaOH. After reduction process by using hydrazine hydrate and Pd/C amine compound (2) was formed. New crown ether imine compounds (3–5) were synthesized by the condensation of corresponding crown ether diamine (2) with salicylaldehyde derivatives. Sodium complexes of the crown compounds (1a–5a) form crystalline 1:1 (Na⁺: ligand) complexes with sodium perchlorate. Nickel(II) complexes (3b–5b) with 1:1 (Ni²⁺:ligand) stoichiometries were also been synthesized from the Schiff bases (3–5). The results indicated that the Schiff base ligands coordinated through the azomethine nitrogen and phenolic oxygen. The extraction ability of compounds (1, 3, 4 and 5) were also evaluated in chloroform by using several alkali and transition metal picrates such as Li⁺, Na⁺, K⁺, Cr³⁺, Mn²⁺, Ni²⁺, Cu²⁺, Zn²⁺ and Pb²⁺.     

Time-resolved fluorescence microscopy for quantitative Ca2+ imaging in living cells

Calcium (Ca²⁺) is a ubiquitous intracellular second messenger and involved in a plethora of cellular processes. Thus, quantification of the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and of its dynamics is required for a comprehensive understanding of physiological processes and potential dysfunctions. A powerful approach for studying [Ca²⁺]_i is the use of fluorescent Ca²⁺ indicators. In addition to the fluorescence intensity as a common recording parameter, the fluorescence lifetime imaging microscopy (FLIM) technique provides access to the fluorescence decay time of the indicator dye. The nanosecond lifetime is mostly independent of variations in dye concentration, allowing more reliable quantification of ion concentrations in biological preparations. In this study, the feasibility of the fluorescent Ca²⁺ indicator Oregon Green Bapta-1 (OGB-1) for two-photon fluorescence lifetime imaging microscopy (2P-FLIM) was evaluated. In aqueous solution, OGB-1 displayed a Ca²⁺-dependent biexponential fluorescence decay behaviour, indicating the presence of a Ca²⁺-free and Ca²⁺-bound dye form. After sufficient dye loading into living cells, an in situ calibration procedure has also unravelled the Ca²⁺-free and Ca²⁺-bound dye forms from a global biexponential fluorescence decay analysis, although the dye's Ca²⁺ sensitivity is reduced. Nevertheless, quantitative [Ca²⁺]_i recordings and its stimulus-induced changes in salivary gland cells could be performed successfully. These results suggest that OGB-1 is suitable for 2P-FLIM measurements, which can gain access to cellular physiology.

Crescent aromatic oligothioamides as highly selective receptors for copper(Ⅱ) ion

A series of crescent aromatic oligothioamides(4, 6, 8, 15, and 18) bearing different number of sulfur atoms were designed and synthesized via thionation of their corresponding aromatic oligoamides(3, 5, and 7) using Lawesson's reagent. The X-ray structure of a trimeric analogue(13) revealed the presence of intramolecular three-center hydrogen bonds that are responsible for the rigidification of the molecular backbone. The extraction by these novel receptors toward some representative heavy metal cations(Zn2+, Cd2+, Co2+, Ni2+, Pb2+, and Cu2+) and alkali and alkaline earth metal cations(Li+, Na+, K+, Rb+, Cs+, Ca2+, and Sr2+) demonstrated high efficiency(83.5%–96.4%) and superior selectivity for Cu2+ over other selected metal cations. Particularly, the extractability was correlated to both the number of sulfur atoms and orientation of thiocarbonyl groups as revealed in the order: 6 4 18 15. This is in stark contrast to the oligoamides that only gave much lower extractability(5.9%–16.4%), suggestive of the importance of replacement of carbonyl oxygen atoms with sulfur atoms in the extraction of Cu2+. The complexation behavior of 4, 6, and 8 with Cu2+ was also examined by UV-Vis and NMR techniques.     

Crown-containing styryl dyes

Complexation of styryl dyes containing theN-phenylaza-15-crown-5 fragment (trans-2a,b) with Ca²⁺ ions in MeCN was studied. Unlike cationic dyetrans-2a, betainetrans-2b forms both complexes of 1∶1 stoichiometry and aggregates consisting of four dye molecules and one Ca²⁺ ion. Cation-induced aggregation was observed also for the analog of dyetrans-2b, which contains a dimethylamino group instead of an azacrown ether fragment (trans-3). Apparently, the interaction between a metal cation and sulfo groups of moleculestrans-2b ortrans-3 makes the main contribution to the stability of aggregates. The dependence of the stability of aggregates on the nature of the metal cation was studied.     

1,2-Dichlorobenzene affects the formation of the phosphoenzyme stage during the catalytic cycle of the Ca<Superscript>2+</Superscript>-ATPase from sarcoplasmic reticulum

Background

1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca²⁺-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca²⁺-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca²⁺ concentration.

Results

Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca²⁺-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca²⁺ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca²⁺ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca²⁺ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-³²P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.

Conclusions

The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E₂P?·?Ca₂ state to E₂ and P_i, which explains the uncoupling of the ATP hydrolysis from the Ca²⁺ transport.

     

Comparison of Ca2+ and Mg2+ binding to calmodulin. An enthalpy,entropy, and gibbs free energy analysis

A consistent set of G _B , H _B , and S _B parameters have been determined from ion specific electrode, calorimetric, and spectrophotometric studies for the binding of Ca²⁺ and Mg²⁺ to bovine calmodulin at pH=7.0 and an ionic strength I of 0.113M. A non-linear least squares analysis of calcium specific ion electrode data yields, on a molar basis, four calcium dissociation constants: 10^–7 for the first site, 10^–5 for the fourth site, and two constants between these values. Both calorimetric experiments and an indicator method provide evidence that Mg²⁺ binds to calmodulin, probably at the same sites as Ca²⁺, but with affinities about 100 times smaller: 4×10^–5 for the first site and 2×10^–3 for the fourth. Calorimetric titrations on Ca²⁺ binding to calmodulin in three buffers are consistent with 0.46 protons released upon binding at all four sites and yield an average H _B per site of 5.6 and 7.9 kJ-mol^–1 for Ca²⁺ and Mg²⁺, respectively. The entropy of the system increases by 524 and 361 J-K^–1-mol^–1 when Ca²⁺ and Mg²⁺, respectively, bind to four sites on calmodulin, i.e., the selectivity of calmodulin for Ca²⁺ is primarily derived from entropy effects. Further analysis based on elimination of the entropy term for the metal ions demonstrates that calmodulin bound to Ca²⁺ has a larger entropy than the unbound calmodulin; the opposite is true for calmodulin bound to Mg²⁺. These analyses are consistent with the hypothesis that Ca²⁺ forms tight complexes at all sites on calmodulin and that release of waters of hydration upon binding is the source of the increase of entropy in the system.     

Synthesis and cation complexation of calix[4]azacrowns and a spirobiscalix[4]azacrown

The synthesis and complexation properties of three new derivatives, one spirobiscalix[4]azacrown (1) and two calix[4]azacrown (2 and 3), are reported. Complexation studies of the three ligands toward transition and heavy metal cations have been carried out and monitored by UV absorption spectrophotometry in acetonitrile. Mononuclear complexes were detected for all complexes, whereas binuclear species (M₂L) were also formed in the case of ligand 1 with Cu²⁺ and Pb²⁺. The extraction properties of 1, 2 and 3 toward Cu²⁺, Zn²⁺, Pb²⁺, Ag²⁺ and Cd²⁺ are also reported. The results showed that complexation is the main factor affecting extraction with ligands 2 and 3, while with ligand 1 it is not.     

Structural insights into calmodulin/adenylyl cyclase 8 interaction

Calmodulin (CaM) is a highly conserved intracellular Ca²⁺-binding protein that exerts important functions in many cellular processes. Prominent examples of CaM-regulated proteins are adenylyl cyclases (ACs), which synthesize cAMP as a central second messenger. The interaction of ACs with CaM represents the link between Ca²⁺-signaling and cAMP-signaling pathways. Thereby, different AC isoforms stimulated by CaM, comprise diverse mechanisms of regulation by the Ca²⁺ sensor. To extend the structural information about the detailed mechanisms underlying the regulation of AC8 by CaM, we employed an integrated approach combining chemical cross-linking and mass spectrometry with two peptides representing the CaM-binding regions of AC8. These experiments reveal that the structures of CaM/AC8 peptide complexes are similar to that of the CaM/skeletal muscle myosin light chain kinase peptide complex where CaM is collapsed around the target peptide that binds to CaM in an antiparallel orientation. Cross-linking experiments were complemented by investigating the binding of AC8 peptides to CaM thermodynamically with isothermal titration calorimetry. There were no hints on a complex, in which both AC8 peptides bind simultaneously to CaM, refining our current understanding of the interaction between CaM and AC8.