43Ca NMR studies of Ca2+-Tetrahymena calmodulin complexes

⁴³Ca NMR spectra of Ca²⁺-Tetrahymena calmodulin(Tet. CaM.) complexes have been observed under various conditions. Off-rate of Ca²⁺ from Tet. CaM. is estimated to be approx. 2.7 × 10³ s^?1 under a certain assumption. Relaxation rates of ⁴³Ca NMR of Ca²⁺-Tet. CaM. are remarkably increased(by one order in magnitude) by adding trifluoperazine(TFP), a potent calmodulin antagonist. Relaxation parameters estimated suggest that Ca²⁺ mobility is reduced by the TFP binding. A stoichiometry of TFP is two moles per Tet. CaM. molecule. The relaxation rates of ⁴³Ca NMR signals are increased by adding excessive Mg²⁺ to the Ca²⁺-Tet. CaM. solutions. The addition of Mg²⁺ to the Ca²⁺-Tet. CaM. complex decreases apparent pKa value of the complex as well.     

Trifluoperazine binding to calmodulin: a shift reagent 43CA NMR study

43Ca NMR experiments of Ca2+ binding to calmodulin (CaM) were performed in the presence and absence of the calmodulin antagonist trifluoperazine (TFP). By making use of the shift reagent Dy(PPP)(7-) (a 1:2 complex of DyCl3 and Na5P3O10) we have succeeded in separating the 43Ca resonances of protein-bound Ca2+ and free Ca2+ in the otherwise unresolved spectra. This experimental strategy has allowed us to demonstrate unequivocally that the affinity of CaM for Ca2+ is markedly increased in the presence of TFP. Thus Ca2+ is not liberated from the protein upon addition of TFP as had been suggested based on earlier 43Ca NMR experiments (Shimuzu, T., Hatano, M., Nagao, S. and Nozawa, Y. (1982), Biochem. Biophys. Res. Comm. 106, 1112-1118).     

Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.     

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes

A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.     

The gating effect of calmodulin and calcium on the connexin50 hemichannel

Gap junction channels formed by connexin50 (Cx50) are critical for the maintenance of eye lens transparency, which is sensitive to pH and external Ca2+ concentration, but the mechanism is still not clear. In this study we performed dye uptake-leakage assays, patch clamping and confocal co-localization experiments to confirm the function of calmodulin (CaM) and Ca2+ in the Cx50 hemichannel. Below pH 7.4, lucifer yellow (LY)-preloaded Cx50-HeLa cells allow dye to leak out when washed with Ca2+-free solution or incubated in solution containing 50 microg/ml W7 (CaM inhibitor) first, then washed in solution containing 2 mM Ca2+, whereas little or no dye leakage was observed when LY-preloaded Cx50-HeLa cells were incubated in solution containing 2 mM Ca2+. Moreover, in the absence of Ca2+, polarizing pulses applied to Cx50-HeLa activated outward transmembrane currents, which were inhibited by 2 mM external Ca2+. When Cx50-HeLa cells were incubated with 2 mM Ca2+ and 50 microg/ml W7, the transmembrane currents were activated again. This indicates that Ca2+ and CaM play a gating role in Cx50 hemichannels. Either the chelation of Ca2+ or the inhibition of CaM increased the permeability of Cx50 hemichannels. The same phenomena were observed below pH 6.5. Furthermore, CaM could be localized in gap junctions formed by Cx50 below pH 6.5. Our results demonstrate that CaM and Ca2+ can cooperate in the gating control of Cx50 hemichannels.     

Site-specific derivatives of wheat germ calmodulin. Interactions with troponin and sarcoplasmic reticulum

Wheat germ calmodulin (CaM) was derivatized at its single cysteine (Cys27) with either the fluorescent reagent, N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (I-EDANS) or the photoactivable cross-linker benzophenone-4-maleimide. Comparison of the native and derivatized wheat germ CaMs with native bovine testis CaM indicates that the concentrations of these proteins required for half-maximal stimulation of either erythrocyte membrane Ca2+-ATPase activity or cardiac sarcoplasmic reticulum phosphorylation are very similar. Affinity labeling of troponin subunits with 125I- and benzophenone-4-maleimide-labeled CaM demonstrates CaM binding to troponin I (TnI) and troponin T (TnT) in binary complexes, as well as to both subunits in the CaM.TnI.TnT ternary complex. This suggests that both subunits are within 10 A of Cys27 of calmodulin. Affinity labeling of cardiac sarcoplasmic reticulum vesicles with 125I- and benzophenone-4-maleimide-labeled CaM exhibits a Ca2+- and Mg2+-dependent labeling of phospholamban, as shown previously with bovine calmodulin (Louis, C.F., and Jarvis, B. (1982) J. Biol. Chem. 257, 15187-15191). Thus, it appears that Ca2+-binding site I of calmodulin is at or near binding sites of calmodulin for TnI, TnT, and phospholamban. Analysis of the time-resolved fluorescence decay curves of I-EDANS-labeled calmodulin indicates a major component with a lifetime of 11.9 ns (+Ca2+), which accounts for 81% of the total fluorescence. The lifetime decreases slightly to 11.3 ns in the absence of Ca2+. Fluorescence anisotropy experiments indicate that I-EDANS-labeled CaM binds TnI with Kd = 6 x 10(-8) M in the presence of Ca2+. This study suggests that these single-site derivatives will be useful for characterizing a variety of calmodulin-receptor interactions because they lack ambiguities inherent in less specific labeling methods.     

Calcium dependence of the interaction between calmodulin and anthrax edema factor

Edema factor (EF), a toxin from Bacillus anthracis (anthrax), possesses adenylyl cyclase activity and requires the ubiquitous Ca2+-sensor calmodulin (CaM) for activity. CaM can exist in three major structural states: an apo state with no Ca2+ bound, a two Ca2+ state with its C-terminal domain Ca2+-loaded, and a four Ca2+ state in which the lower Ca2+ affinity N-terminal domain is also ligated. Here, the interaction of EF with the three Ca2+ states of CaM has been examined by NMR spectroscopy and changes in the Ca2+ affinity of CaM in the presence of EF have been determined by flow dialysis. Backbone chemical shift perturbations of CaM show that EF interacts weakly with the N-terminal domain of apoCaM. The C-terminal CaM domain only engages in the interaction upon Ca2+ ligation, rendering the overall interaction much tighter. In the presence of EF, the C-terminal domain binds Ca2+ with higher affinity, but loses binding cooperativity, whereas the N-terminal domain exhibits strongly reduced Ca2+ affinity. As judged by chemical shift differences, the N-terminal CaM domain remains bound to EF upon subsequent Ca2+ ligation. This Ca2+ dependence of the EF-CaM interaction differs from that observed for most other CaM targets, which normally interact only with the Ca2+-bound CaM domains and become active following the transition to the four Ca2+ state.     

Interaction of a fluorescent N-dansylaziridine derivative of troponin I with calmodulin in the absence and presence of calcium

Rabbit skeletal muscle troponin I was covalently labeled with N-dansylaziridine, resulting in a fluorescent labeled protein. This derivative (DANZTnI) and native troponin I (TnI) inhibited calmodulin (CaM) stimulation of bovine heart Ca2+-sensitive cyclic nucleodite phosphodiesterase with identical inhibition constants. Association of DANZTnI with calmodulin was monitored directly by changes in flourescence intensity in the presence of Ca2+ and by changes in fluorescence anisotropy in the absence of Ca2+. Quantitation of the affinity of calmodulin for calmodulin-binding proteins in both the presence and absence of Ca2+ is necessary for prediction of the extent of interaction of both Ca2+ and calmodulin-binding proteins with calmodulin in vivo. The dissociation constants for the DANZTnI-calmodulin-l4Ca2+ and DANZTnI-calmodulin complexes were 20 nM and 70 micrometers, respectively. These dissociation constants define a free energy coupling of-4.84 kcal/mol of troponin I for binding of Ca2+ and troponin I to calmodulin. The Ca2+ dependence for troponin I-calmodulin complex formation predicted from these experimentally determined parameters was closely approximated by the Ca2+ dependence for complex formation between troponin I and fluorescent 5-[[[(iodoacetyl)amino]ethyl]-amino]-1-napthalenesulfonic acid derivatized calmodulin as determined by fluorescence anisotropy. Complex formation occurred over a relatively narrow range of Ca2+ concentration, indicative of positive heterotropic cooperativity for Ca2+ and troponin I binding to calmodulin.     

Crystal structures of apocalmodulin and an apocalmodulin/SK potassium channel gating domain complex

Small conductance Ca2+-activated K+ channels (SK channels) are composed of the pore-forming alpha subunit and calmodulin (CaM). CaM binds to a region of the alpha subunit called the CaM binding domain (CaMBD), located intracellular and immediately C-terminal to the inner helix gate, in either the presence or absence of Ca2+. SK gating occurs when Ca2+ binds the N lobe of CaM thereby transmitting the signal to the attached inner helix gate to open. Here we present crystal structures of apoCaM and apoCaM/SK2 CaMBD complex. Several apoCaM crystal forms with multiple (12) packing environments reveal the same EF hand domain-swapped dimer providing potentially new insight into CaM regulation. The apoCaM/SK2 CaMBD structure, combined with our Ca2+/CaM/CaMBD structure suggests that Ca2+ binding induces folding and dimerization of the CaMBD, which causes large CaMBD-CaM C lobe conformational changes, including a >90 degrees rotation of the region of the CaMBD directly connected to the gate.     

A new role for IQ motif proteins in regulating calmodulin function

IQ motifs are found in diverse families of calmodulin (CaM)-binding proteins. Some of these, like PEP-19 and RC3, are highly abundant in neuronal tissues, but being devoid of catalytic activity, their biological roles are not understood. We hypothesized that these IQ motif proteins might have unique effects on the Ca2+ binding properties of CaM, since they bind to CaM in the presence or absence of Ca2+. Here we show that PEP-19 accelerates by 40 to 50-fold both the slow association and dissociation of Ca2+ from the C-domain of free CaM, and we identify the sites of interaction between CaM and PEP-19 using NMR. Importantly, we demonstrate that PEP-19 can also increase the rate of dissociation of Ca2+ from CaM when bound to intact CaM-dependent protein kinase II. Thus, PEP-19, and presumably similar members of the IQ family of proteins, has the potential to alter the Ca2+-binding dynamics of free CaM and CaM that is bound to other target proteins. Since Ca2+ binding to the C-domain of CaM is the rate-limiting step for activation of CaM-dependent enzymes, the data reveal a new concept of importance in understanding the temporal dynamics of Ca2+-dependent cell signaling.     

Purification of recombinant proteins based on the interaction between a phenothiazine-derivatized column and a calmodulin fusion tail.

A method to purify proteins by fusing them to the Ca2+-dependent protein calmodulin is described by using glutathione-S-transferase (GST) from Schistosoma japonicum as a model. Glutathione-S-transferase was genetically fused to calmodulin (CaM). The designed GST-CaM fusion protein has a selective factor Xa cleavage site located between the C-terminus of GST and the N-terminus of CaM. The recombinant fusion protein was expressed in Escherichia coli, and the crude cell extract was loaded onto a phenothiazine affinity column in the presence of Ca2+. Calmodulin was used as an affinity tail to enable binding of the fusion protein to the phenothiazine column. Removal of Ca2+ with a calcium-complexing solution causes elution of the fusion protein. The GST-CaM fusion protein was then digested with factor Xa, and the target protein GST was isolated. The purity of the isolated GST was verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).     

马兜铃酸与钙调素相互作用的荧光光谱分析——一种非钙离子依赖性钙调素拮抗剂的研究

本文报道我室近来发现的一种天然钙调素(Calmodulin,CaM)拮抗剂马兜铃酸(Aristolochic acid,ATA)的研究。利用丹磺酰标记的CaM(D-CaM)对马兜铃酸的研究表明,马兜铃酸是一种非钙离子依赖性钙调素拮抗剂,实验测得马兜铃酸与D-CaM结合的解离常数,有Ca~(2+)和无Ca~(2+)情况下分别为70μmol/L、77μmol/L。两种状况下马兜铃酸对D-CaM荧光强度的抑制分别为40％、41％。暗示马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区之外。三氟啦嗪(TFP)引起的D-CaM荧光增强可被马兜铃酸明显降低,而TFP在达到马兜铃酸浓度的15倍以上仍未能逆转马兜铃酸对D-CaM荧光强度的降低作用,这为马兜铃酸主要作用于CaM上Ca~(2+)诱导的疏水区以外提供了又一佐证。     

GM_1激活环核苷酸磷酸二酯酶的作用

神经节苷脂GM1能激活CaM依赖性环核苷酸磷酸二酯酶,其AC50为1．56μg／ml．这种作用并不一定依赖Ca2＋的存在．在有Ca2＋存在时,GM1对PDE的最大激活活性低于CaM,仅为CaM的80．4％;没有Ca2＋存在时,GM1与CaM对PDE有同样的最大激活活性（100％）．GM1能使CaM对PDE的激活曲线左移,AC50降低,但不改变CaM对PDE的最大激活活性．三氟啦嗪能使GM1激活的PDE的活性降低,其IC50为16．3μmol／L．     

Evidence for the Binding of Calmodulin to Endogenous B-50 (GAP-43) in Native Synaptosomal Plasma Membranes

The neuron-specific protein B-50 has been described as an atypical calmodulin (CaM) binding protein, because the purified protein has a higher affinity for CaM in the absence than in the presence of Ca2+. We have studied CaM binding to endogenous B-50 in native synaptosomal plasma membranes (SPM) and growth cone membranes in order to assess the physiological relevance of the binding. To detect B-50/CaM binding, we used the cross-linker disuccimidyl suberate (DSS) to form a covalent B-50/CaM complex, which is stable on SDS-PAGE. Upon addition of DSS, purified B-50 and calmodulin form a 70-kDa complex in the absence but not in the presence of Ca2+. This complex can be detected by protein staining and on Western blots using anti-B-50 and anti-CaM IgGs. DSS treatment of SPM or growth cone membranes with or without exogenous CaM results in the formation of a 70-kDa B-50/CAM complex detectable only in the absence of Ca2+ with both antibodies. Our results strongly suggest that the binding of CaM to endogenous B-50 in SPM and growth cone membranes is of physiological relevance. CaM binding to B-50 may be an important factor in regulating neurite outgrowth and/or neurotransmitter release.     

Calmodulin activates inositol 1,4,5-trisphosphate 3-kinase activity in pig aortic smooth muscle.

The effects of calmodulin (CaM) on inositol 1,4,5-trisphosphate (InsP3) 3-kinase activity in pig aortic smooth muscle were examined. The cytosol fraction of muscle cells, containing 1.2-2.0 micrograms of CaM/mg of cytosol protein (thus 0.12-0.2%, w/w), showed a Ca2+-dependent InsP3 3-kinase activity, and there was no further activation by exogenous addition of CaM purified from dog brain. (NH4)2SO4 fractionation of the cytosol fraction revealed that a 20-60%-satd.-(NH4)2SO4 fraction was rich in the enzyme activity, and the activity without exogenous CaM was still dependent on Ca2+, although the CaM content in this fraction was minute (0.013-0.016%, w/w). The kinase activity observed in the absence of exogenous CaM became insensitive to Ca2+ when a 20-60%-satd.-(NH4)2SO4 fraction was applied to a DEAE-cellulose column, but exogenous addition of CaM increased the enzyme activity from 80-120 to 450 pmol/min per mg of protein, with addition of 10 microM free Ca2+. A fraction separated by DEAE-cellulose chromatography was applied to a CaM affinity column. The kinase activity was retained on the column in the presence of Ca2+, and was eluted by lowering the free Ca2+ concentration by adding EGTA. These results directly show that CaM activates InsP3 3-kinase activity and the enzyme becomes sensitive to Ca2+.     

Trifluoperazine binding to porcine brain calmodulin and skeletal muscle troponin C

Binding of trifluoperazine (TFP), a phenothiazine tranquilizer, to porcine brain calmodulin (CaM) and rabbit skeletal muscle troponin C (Tn C) was measured by an automated high-performance liquid chromatography binding assay using a molecular sieving column; 10 micrograms of either protein per injection is sufficient for determining TFP binding, and results are comparable to those obtained by equilibrium dialysis. Very little binding was observed to either protein in the absence of Ca2+ while in the presence of Ca2+ both proteins bind 4 equiv of TFP. Other characteristics of TFP binding however are different for each protein. For CaM, half-maximal binding occurs at 5.8 microM TFP, the Hill coefficient is 0.82, and the fit of the data to the Scatchard equation is consistent with four independent TFP-binding sites. Binding of one melittin displaces two TFP from CaM. Thus, there are two recognizable classes of TFP-binding sites: those that are displaced by melittin and those that are not. TFP causes an increase in the Ca2+ affinity of CaM, and three Ca2+ must be bound to CaM for TFP binding to occur. The studies also yielded a measure of the intrinsic affinity of three of CaM's Ca2(+)-binding sites that is in agreement with previous reports. For troponin C, half-maximal binding occurs at 16 microM TFP, the Hill coefficient is 1.7, and the data best fit the Adair equation for four binding sites. The measured constants K1, K2, K3, and K4 were 2.5 X 10(4), 6.6 X 10(3), 5.8 X 10(5), and 2.0 X 10(5) M-1, respectively, in 1 mM Ca2+ and were similar when Mg2+ was additionally included. TFP also increases troponin C's Ca2+ affinity, and it is the low-affinity, Ca2(+)-specific binding sites that are affected. These studies yielded a measure of the intrinsic affinity of these Ca2(+)-binding sites that is in agreement with previous measurements.     

The two IQ-motifs and Ca2+/calmodulin regulate the rat myosin 1d ATPase activity

The light chain binding domain of rat myosin 1d consists of two IQ-motifs, both of which bind the light chain calmodulin (CaM). To analyze the Myo1d ATPase activity as a function of the IQ-motifs and Ca2+/CaM binding, we expressed and affinity purified the Myo1d constructs Myo1d-head, Myo1d-IQ1, Myo1d-IQ1.2, Myo1d-IQ2 and Myo1dDeltaLV-IQ2. IQ1 exhibited a high affinity for CaM both in the absence and presence of free Ca2+. IQ2 had a lower affinity for CaM in the absence of Ca2+ than in the presence of Ca2+. The actin-activated ATPase activity of Myo1d was approximately 75% inhibited by Ca2+-binding to CaM. This inhibition was observed irrespective of whether IQ1, IQ2 or both IQ1 and IQ2 were fused to the head. Based on the measured Ca2+-dependence, we propose that Ca2+-binding to the C-terminal pair of high affinity sites in CaM inhibits the Myo1d actin-activated ATPase activity. This inhibition was due to a conformational change of the C-terminal lobe of CaM remaining bound to the IQ-motif(s). Interestingly, a similar but Ca2+-independent inhibition of Myo1d actin-activated ATPase activity was observed when IQ2, fused directly to the Myo1d-head, was rotated through 200 degrees by the deletion of two amino acids in the lever arm alpha-helix N-terminal to the IQ-motif.     

The regulation of Ca2+ transport by fast skeletal muscle sarcoplasmic reticulum. Role of calmodulin and of the 53,000-dalton glycoprotein

Ca2+ uptake and Ca2+-dependent ATP hydrolysis of fast skeletal muscle sarcoplasmic reticulum (SR) are strongly inhibited by trifluoperazine (TFP). Inhibition, which is Ca2+-dependent, is 90% with 14 microM TFP and 0.2 microM Ca2+. TFP interacts strongly, in a Ca2+-dependent way, with two SR proteins, calmodulin and the 53,000-dalton glycoprotein. The two proteins were purified by TFP affinity chromatography. The inhibition of SR activity by TFP was correlated with the interaction of the drug with the glycoprotein, rather than with calmodulin. The main effect was a shift of the (Ca2+-Mg2+)-ATPase from a high to a low affinity form. Calmodulin-dependent phosphorylation of three proteins (Mr = 57,000, 35,000, and 20,000) of the SR membrane of fast skeletal muscle was also demonstrated. Phosphorylation of these three proteins plays no role in the regulation of the active Ca2+-uptake reaction.     

Effects of Ca2+ and Zn2+ on trifluoperazine-S100 proteins interactions: induced circular dichroism and fluorescence spectra

Interactions of trifluoperazine (TFP) with S100 proteins, EF-hand type Ca2+-binding proteins, in the presence of Ca2+ and Zn2+ were studied with induced circular dichroism (CD) and fluorescence spectra. The positive CD bands of TFP were induced at around 265 nm by adding either S100a or S100a0 protein in the presence of Ca2+. No CD band of TFP was, however, induced by adding S100b protein in the presence of Ca2+. Addition of Zn2+ to the TFP/S100 protein solutions did not induce any CD band at all. The fluorescence intensity of 2-p-toluidinylnaphthalene 6-sulfonate (TNS) bound to S100a or S100a0 protein decreased by adding TFP in the presence of Ca2+, while that bound to S100b protein decreased by adding TFP in the presence of Zn2+, indicating that TFP binds to S100a protein and S100a0 protein in a Ca2+-dependent manner and to S100b protein in a Zn2+-dependent manner. From these results together with other experimental findings it was suggested that (1) TFP binds to S100a protein and S100a0 protein in the presence of Ca2+, with half-saturation points of 18 and 3 microM, respectively, (2) TFP binds to S100b protein only in the presence of Zn2+, (3) alpha-subunit of S100 protein binds to TFP specifically in a Ca2+-dependent manner and beta-subunit in a Zn2+-dependent manner.     

Backbone and side chain dynamics of mutant calmodulin-peptide complexes

The mechanism of long-range coupling of allosteric sites in calcium-saturated calmodulin (CaM) has been explored by characterizing structural and dynamics effects of mutants of calmodulin in complex with a peptide corresponding to the smooth muscle myosin light chain kinase calmodulin-binding domain (smMLCKp). Four CaM mutants were examined: D95N and D58N, located in Ca2+-binding loops; and M124L and E84K, located in the target domain-binding site of CaM. Three of these mutants have altered allosteric coupling either between Ca2+-binding sites (D58N and D95N) or between the target- and Ca2+-binding sites (E84K). The structure and dynamics of the mutant calmodulins in complex with smMLCKp were characterized using solution NMR. Analysis of chemical shift perturbations was employed to detect largely structural perturbations. 15N and 2H relaxation was employed to detect perturbations of the dynamics of the backbone and methyl-bearing side chains of calmodulin. The least median squares method was found to be robust in the detection of perturbed sites. The main chain dynamics of calmodulin are found to be largely unresponsive to the mutations. Three mutants show significantly perturbed dynamics of methyl-bearing side chains. Despite the pseudosymmetric location of Ca2+-binding loop mutations D58N and D95N, the dynamic response of CaM is asymmetric, producing long-range perturbation in D58N and almost none in D95N. The mutations located at the target domain-binding site have quite different effects. For M124L, a local perturbation of the methyl dynamics is observed, while the E84K mutation produces a long-range propagation of dynamic perturbations along the target domain-binding site.