Conformation Of Calmodulin Research Articles

Functional conformations of calmodulin: I. Preparation and characterization of a conformational specific anti-bovine calmodulin monoclonal antibody.

Calmodulin, similarly to many other Ca(2+)-activated proteins, undergoes considerable conformational changes in the presence of Ca2+ ions. These changes were followed using specific monoclonal antibodies against calmodulin. Since calmodulin is a poor immunogen due to its high phylogenetic conservancy, glutaraldehyde-crosslinked bovine brain extract, which contains a considerable amount of functionally active calmodulin complexed with its target proteins, was used as an antigen. Out of nine anti-calmodulin mAbs isolated, three (namely, CAM1, CAM2 and CAM4) were purified and characterized. MAb CAM1 was identified as an IgG1 while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAM1 binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAM1 was found to be especially sensitive to the conformational state of calmodulin in the presence of Ca2+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca2+ removal. In addition mAb CAM1 failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM1 and CAM4 share common epitopes with the above molecules.

Two-dimensional NMR Studies of Selenomethionyl Calmodulin

Calmodulin (CaM) is a ubiquitous calcium regulatory protein that can interact with almost 30 different target proteins. The majority of the CaM-binding domains of the target proteins are believed to interact with two hydrophobic surfaces on Ca2+-CaM; these two regions are very rich in Met residues. To obtain more information about the role of these residues, we have biosynthetically incorporated selenomethionine (SeMet) in place of the nine Met residues of CaM. Amino acid analysis shows that the SeMet-CaM contains 15% Met and 85% SeMet. SeMet-CaM retains many of the properties of the wild-type protein; it activates the enzyme cyclic nucleotide phosphodiesterase, it binds to phenyl-Sepharose and myosin light chain kinase (MLCK) in a calcium-dependent manner, and it experience a calcium-dependent band shift during SDS-gel electrophoresis. Moreover, by comparing the natural abundance (1H, 13C)-heteronuclear multiple quantum coherence (HMQC) spectra of the calcium, apo and target peptide-bound forms of wild-type CaM and SeMet-CaM, we have found that the two proteins have very similar, if not identical, structures. Thus, the substitution of SeMet for Met does not cause a change in the conformation and function of CaM, in agreement with the results obtained for other proteins. The apo, calcium and target peptide-bound forms of SeMet-CaM were subsequently studied by natural abundance (1H, 77 Se)-heteronuclear multiple bond correlation (HMBC) and (1H, 13C)-HMQC NMR. Nine well-resolved 77Se resonances could be observed. Substitution of SeMet for Met gave rise to the same 1H and 13C chemical shift changes for each individual Met residue, this facilitated making the assignments from known 1 H, 13C assignments of the Met residues. Some of these assignments were confirmed by studying Met→Leu mutants of CaM. With the exception of Met76, which always remains solvent exposed, all resonances experienced large 77Se chemical shift changes upon the addition of Ca2+ and the MLCK peptide. The large shift changes indicate that the electron distribution in the SeMet side-chain can be adjusted for the different states of CaM, suggesting that the polarizability of sulfur or selenium may be important for the proper functioning of CaM. This study also shows that the natural abundance (1H, 77Se)-HMBC experiment provides a sensitive approach for the study of SeMet protein

Alteration of calmodulin-protein interactions by a monoclonal antibody to calmodulin

The effects of specific anti-calmodulin monoclonal antibodies on the conformation and interaction of calmodulin with two enzymes, the insulin receptor tyrosine kinase and casein kinase II, are examined. Addition of the anti-calmodulin antibody 2D1 in vitro augments phosphorylation of calmodulin by rat hepatocyte insulin receptors 4.9 ± 0.5-fold ( n = 7). Nonimmune immunoglobulin has no effect. Maximal phosphorylation is observed at a molar ratio of calmodulin: antibody of approx. 2: 1, with higher concentrations of antibody producing lesser enhancement. Increasing Ca 2+ concentrations in the physiological range progressively inhibit phosphorylation both in the absence and presence of antibody 2D1. Phosphate is incorporated predominantly on Tyr-99, which is distant from the antibody binding site. Enhancement of casein kinase II-catalyzed calmodulin phosphorylation is also produced by the antibody 2D1, implying that antibody binding induces a change in calmodulin conformation. In contrast, two other anti-calmodulin monoclonal antibodies, 4F4 and 4G2, decrease phosphorylation of calmodulin by both the insulin receptor kinase and casein kinase II. These data indicate that secondary and tertiary structures are important in enzyme-substrate interactions and suggest that the antibodies may be useful in investigating the mechanism of calmodulin function.

The role of the autoinhibitory domain in differential metal ion activation of calmodulin-stimulated phosphatase

Metal ion activators, Ni 2+ and Mn 2+, have been suggested to induce different conformations of calmodulin (CaM)-stimulated phosphatase. In the present study, an autoinhibitory domain previously implicated in the conformation transition of CaM stimulation of the phosphatase is shown to participate in defining the differential metal ion activation. A proteolytic derivative of the phosphatase deleted from the autoinhibitory domain displayed CaM-independent Mn 2+-stimulated activity which was about 4-times that of the CaM-stimulated activity of the native enzyme. The Ni 2+-stimulated activity of the derivative, on the other hand, retained slight CaM-dependence, and the CaM-stimulated activity was 90% of that of the native enzyme. A synthetic peptide corresponding to the autoinhibitory domain could inhibit the Mn 2+-stimulated activity of the phosphatase derivative by 80%, but had little effect on the Ni 2+-stimulated activity.

Spin-labeling studies of the conformation of the Ca(2+)-regulatory protein calmodulin in solution and bound to the membrane skeleton in erythrocyte ghosts: implications to transmembrane signaling

Electron paramagnetic resonance (EPR) studies of the Ca(2+)-regulatory protein calmodulin (CaM) have been performed. The conformation of CaM in solution changes upon binding of Ca2+ allowing the protein to bind to target proteins existing in the red blood cell membrane. In this study a maleimide spin label, covalently attached to the single cysteine residue of CaM located in the first Ca(2+)-binding domain, was used to monitor allosteric conformational changes induced by interaction of CaM with Ca2+ and subsequently with the red blood cell membrane. The results show, relative to apo-CaM, a significant increase in the apparent rotational correlation time, tau, of the spin label when Ca2+ was present in solution (P less than 0.001). When apo-CaM exposed to red blood cell membrane ghosts in the absence of Ca2+, no significant difference in spin label motion was seen relative to solution, consistent with the idea that Ca2+ is required for CaM to bind to skeletal proteins. When Ca2+ was added to CaM which was then exposed to ghosts, a highly significant increase in tau (decrease in motion) (P less than 0.000001) relative to apo-CaM exposed to ghosts was found. This latter increase in tau is significantly greater than that produced by the addition of Ca2+ to CaM in solution (P less than 0.001). The major interaction sites of CaM were found by photoaffinity labeling and autoradiography on SDS-PAGE to be on the principal skeletal protein, spectrin. EPR was also used to investigate the biophysical correlates of transmembrane signaling. Spin-labeled CaM was bound to the membrane skeleton in the presence of Ca2+. On the opposite side of the erythrocyte membrane a lectin was bound to the external glycoconjugate of Band 3, the major transmembrane protein of the erythrocyte. A highly significant increase in T of the maleimide spin probe was found relative to the control system in which the lectin was absent. (P < 0.00001). These results suggest that electron paramagnetic resonance spectra of spin-labeled CaM can provide useful information about protein structure and function when in solution and when bound to membranes.

Construction and molecular dynamics simulation of calmodulin in the extended and in a bent conformation.

Analysis of sequence similarity and comparison of the three-dimensional (3D) structures of troponin C and calmodulin have revealed a sequence in the central helix of calmodulin with a high probability for bending. The three amino acids known to form a bend in the N-terminal portion of troponin C are also found in the central helix of calmodulin. The modelling of a bent calmodulin structure, using the dihedral angles of the three residues in the bend of troponin C as a 3D template, results in a conformation of calmodulin where the N- and C-terminal domains are able to form contacts. Dynamics simulations starting from the X-ray structure of calmodulin and from the modelled bent calmodulin were carried out to compare flexibility and correlated movements of Ca2+ in the binding loops. Both conformations of calmodulin remained stable during the period of simulation. In the simulation of calmodulin in the extended form, the motions of the Ca2+ atoms in the two domains (Ca2+1 and Ca2+2 in one domain, and Ca2+3 and Ca2+4 in the other) are correlated. In the simulation of the bent form, an additional correlation between the Ca atoms in the two different domains is observed. The results are compatible with the occurrence of a bent conformation of calmodulin in the presence of targets, and with increased Ca2+ affinity and cooperativity of the Ca(2+)-binding loops in the calmodulin-peptide complexes.

In vitro effects of organophosphorus compounds on calmodulin activity

In vitro effects of organophosphorus compounds (OP), such as malathion (M), methyl parathion (MP) and ethyl parathion (EP), on calmodulin (CaM) activity and its active conformation were studied to understand the mechanism(s) of neurotoxicity, since CaM is known to regulate Ca2+ transport and the enzymes involved in signal transduction and nucleotide metabolism. The biological activity of CaM was assessed as a measure of phosphodiesterase (PDE) stimulation. The effect of OP compounds on the active conformation of CaM was determined by studying the binding of fluorescence probes, namely N-phenyl-1-naphthylamine (NPN), and changes in dansyl-calmodulin fluorescence. Dansylated calmodulin was also used to study the effect of OP compounds on complex formation between CaM and PDE. All three OP compounds inhibited the CaM activity and its active conformation in a concentration-dependent manner. Malathion was less effective in comparison to EP and MP, with IC50 values of 37 microM, 34.5 microM and 32 microM, respectively, for CaM activity. EP and MP significantly altered NPN and dansyl-calmodulin fluorescence (50 microM concentrations of OP compounds), whereas M did not show any significant effect on NPN fluorescence. All these compounds significantly affected complex formation between the dansylated CaM and PDE. These results suggest that OP compounds may be interacting with CaM, altering its active conformation, and thus may be inhibiting its biological activity.

Calcium-responsive extensible monolayer membrane of calmodulin-albumin conjugate

A Ca(2+)-responsive monolayer protein membrane was prepared by developing calmodulin and bovine serum albumin at the air-water interface and by conjugating them with a bifunctional agent. In the case of the BSA monolayer, complex formation with Mg2+ generated a larger change in surface pressure than that with Ca2+. On the other hand, a drastic change in surface pressure was observed for the conjugated thin membrane associated with Ca2+ than that associated with Mg2+. Due to a drastic change in the conformation of calmodulin, the conjugated protein film changes its morphology (STM image), depending on Ca2+ concentration: the extended structure in the presence of Ca2+ transforms to a shrinked structure in the absence of Ca2+. The largest surface pressure change was detected when calmodulin was mixed with an equimolar amount of bovine serum albumin.

Effect of Organochlorine and Organotin Compounds on Active Conformation of Calmodulin

Abstract The interaction of organotin and organochlorine compounds with calmodulin was studied in order to elucidate whether these compounds interact with calmodulin thereby affecting its active conformation. The conformational changes in calmodulin were studied by using N‐phenyl‐1‐naphthylamine (NPN) and dansyl‐calmodulin as fluorescent probes. Chlordecone, an organochlorine compound, suppressed the increase in fluorescence of NPN by complex formation with calmodulin in the presence of Ca2+ and the effect was concentration dependent with IC50 of 9.25 μM. However, no effect was observed in the presence of 1 mM EGTA. Other organochlorine compounds such as aldrin, dieldrin, endrin and isodrin even at high concentrations (80 μM) did not affect the Ca2+‐dependent change in calmodulin conformation. Chlordecone (20 μM) also inhibited the Ca2+‐dependent increase in dansyl‐calmodulin fluorescence. This chlordecone‐induced, conformationally changed calmodulin (involving the high affinity Ca2+‐binding sites) was un...

A Site-Directed Mutagenesis Study of Yeast Calmodulin1

A site-directed mutagenesis study was carried out in order to understand the regulatory mechanism of calmodulin. We started from the yeast (Saccharomyces cerevisiae) calmodulin gene since it has many differences in amino acid sequence and inferior functional properties compared with the vertebrate calmodulin. Recombinant yeast calmodulins were generated in Escherichia coli transformed by constructed expression plasmids. Three recombinant calmodulins were obtained. The first two were YCM61G, in which the Ca2(+)-binding site 2 (the four Ca2(+)-binding EF-hand structures in calmodulin were numbered from the N-terminus) was converted to the same as that in vertebrate calmodulin, and YCM delta 132-148, in which the C-terminal half sequence of site 4 was deleted. These two recombinant calmodulins had the same maximum Ca2+ binding (3 mol/mol) as yeast calmodulin, which indicates that site 4 of yeast calmodulin was the one losing Ca2+ binding capacity. YCM delta 132-148 could not activate target enzymes, whereas its Ca2+ binding profile was similar to those of yeast calmodulin and YCM61G. Therefore, the structure in site 4 which cannot bind Ca2+ is indispensable for the regulatory function of yeast calmodulin. The complete regulatory function of vertebrate calmodulin can be attained by the combination of 4 Ca2+ binding structures. The negative charge cluster in the central alpha-helix region is suggested to stabilize the active conformation of calmodulin, since the third yeast calmodulin mutant, YCM83E, which had the negative charge cluster, increased the maximum activation of myosin light chain kinase.

Small-angle scattering studies show distinct conformations of calmodulin in its complexes with two peptides based on the regulatory domain of the catalytic subunit of phosphorylase kinase

Small-angle X-ray and neutron scattering have been used to study the solution structures of calmodulin complexed with synthetic peptides corresponding to residues 342-366 and 301-326, designated PhK5 and PhK13, respectively, in the regulatory domain of the catalytic subunit of skeletal muscle phosphorylase kinase. The scattering data show that binding of PhK5 to calmodulin induces a dramatic contraction of calmodulin, similar to that previously observed when calmodulin is complexed with the calmodulin-binding domain peptide from rabbit skeletal muscle myosin light chain kinase. In contrast, calmodulin remains extended upon binding PhK13. In the presence of both peptides, calmodulin also remains extended. Apparently, the presence of PhK13 inhibits calmodulin from undergoing the PhK5-induced contraction. These data indicate that there is a fundamentally different type of calmodulin-target enzyme interaction in the case of the catalytic subunit of phosphorylase kinase compared with that for myosin light chain kinase.

Calmodulin activation of target enzymes. Consequences of deletions in the central helix.

The central helical region of calmodulin (CaM) includes amino acids 65-92 and serves to separate the two pairs of Ca2(+)-binding sites. This region may impart conformational flexibility and also interact with target proteins. The functional effects of deleting two, three, five, or eight amino acids from the central helix were monitored by examining the activation of phosphodiesterase, smooth muscle myosin light chain (MLC) kinase, and Ca2+/CaM-dependent protein kinase II (CaM kinase II). CaMDM(-8), a calmodulin-deletion mutant with 8 amino acids deleted from the middle of the central helix, failed to activate MLC kinase, phosphodiesterase, or CaM kinase II at physiologically significant concentrations of activator but also had altered electrophoretic mobility and tyrosine fluorescence properties suggesting major changes in the structure of this mutant. Deletion of five amino acids (77-81) resulted in an increase in apparent Kact for phosphodiesterase (150-fold), CaM kinase II (25-fold), and MLC kinase (5-fold) relative to CaM. The maximal autophosphorylation activity of CaM kinase II was also diminished 70% with CaMDM(-5). For phosphodiesterase activation, CaMDM(-2) has a 15-fold increase in apparent Kact while CaMDM(-3) had an apparent Kact value only 3-fold higher than native CaM. In contrast, the activation of MLC kinase by the two (79-80)- and three (79-81)-amino acid deletion mutants were indistinguishable from each other or native CaM. CaMDM(-2) and CaMDM(-3) stimulated CaM kinase II autophosphorylation to 85 and 70%, respectively, of native CaM with less than a 2-fold increase in Kact. Therefore, all deletions in the central helix of CaM reduce the efficiency of phosphodiesterase activation as reflected by substantial alterations in Kact. MLC kinase activation, however, is relatively insensitive to small two or three amino acid deletions. CaM kinase II interacts with the central helix deletion mutants in a complex manner with alterations in both the Kact and the maximum activity. The data suggest the central helix of CaM may serve as a flexible tether for MLC kinase (and to a lesser extent CaM kinase II) but that an extended conformation of CaM, as predicted from the crystal structure, may be required for phosphodiesterase activation.

Interaction between lipids and bovine brain calmodulin: lysophosphatidylcholine-induced conformation change

We have monitored the interaction of several lipids with the bovine brain calmodulin(CaM) and analyzed the effect of lysophosphatidycholine (lyso-PC, 2–50 μ g/ml) on conformation of CaM and the interaction between CaM and CaM-binding protein (CaMBP), using a fluorescence signal of 1-(dimethylamino) naphthalene-5-sulfonate-labeled CaM(DNS-CaM). Lyso-PC (egg, 20 μg/ml), among various natural lipids including phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylethanolamine (PE) and their lyso forms, greatly and dose-dependently enhanced the intensity of DNS fluorescence of DNS-CaM in the presence (100 μ M CaCl 2) and absence (1 mM EGTA) of Ca 2+. Apparent dissociation constants calculated from the fluorometric titrations of binding of lyso-PC to DNS-CaM were 0.6 and 3.7 μg/ml in the presence and absence of Ca 2+, respectively. Lyso-PC remarkably prevented both trypsin-induced quenching of the fluorescence of DNS-CaM and tryptic digestion of native CaM in the absence of Ca 2+. Enhancement of DNS fluorescence of DNS-CaM was CaMBP was observed only in the presence of Ca 2+and lyso-PC could further increase the fluorescence intensity of the complex. These all results suggest that lyso-PC can modulate the interaction between CaM and CaMBP as a result of its direct effect on conformation of CaM.

Binding of both Ca2+ and mastoparan to calmodulin induces a large change in the tertiary structure.

The technique of small-angle X-ray scattering has been employed to examine the solution conformation of calmodulin and its complexes with Ca2+ alone, and with both Ca2+ and mastoparan. The radius of gyration decreased by 3.1 +/- 0.3 A upon binding of both 4 mol Ca2+/mol of protein and 1 mol mastoparan/mol of protein to form the ternary complex. A smaller increase was found for the separate binding of 4 mol Ca2+/mol of protein in the absence of mastoparan (0.6 +/- 0.3 A). The analyses of pair distance distribution function showed that the maximal pair distance in calmodulin complex with both Ca2+ and mastoparan decreased by 20-30% in comparison with calmodulin or its complex with Ca2+, and a shoulder near 40 A, which characterizes the dumbbell-shaped molecule of calmodulin, disappeared. These results indicate that the two globular domains of the calmodulin complex with Ca2+ and mastoparan come close together by 8.0-9.5 A on average, if the size and the overall shape of the globular domains are the same in Ca2+-calmodulin-mastoparan complex as in calmodulin or Ca2+-calmodulin complex.

Effects of calcium and calcium analogs on calmodulin: a Fourier transform infrared and electron spin resonance investigation

Fourier transform infrared (FTIR) and electron spin resonance (ESR) spectroscopies have been used to monitor changes in the conformation of calmodulin induced by Ca 2+ and Ca 2+ analogs. Using FTIR spectroscopy we observe that Ca 2+: (i) favors the α-helical conformation and decreases the flexibility of the molecule; (ii) multiplies the intramolecular hydrogen bonds (the ratio of freely vibrating NH/hydrogen bound NH groups decreases); (iii) induces changes in the C-terminal tyrosine environment; and (iv) increases compactness of the molecule (less NH groups in the peptide bonds can be deuterated). As proved by ESR, Ca 2+ binding induces exposure of hydrophobic domains allowing binding of a spin-labelled phenothiazine on calmodulin. When the experiments are performed in the presence of increasing amounts of Ca 2+, both ESR and FTIR provide evidence that major conformational changes result after the filling of only two Ca 2+-binding sites. But achievement of the spectroscopical changes is only observed when the four binding sites are filled (Ca 2+/calmodulin = 4). The effects of analogs are monitored with the same spectroscopical parameters. Zn + does not induce structural modifications of calmodulin but all other analogs studied mimic the calcium effects to some extent. As regards the amplitude of the spectroscopical effects, analogs rank in the following order: Ca 2+ > Cd 2+ > Tb 3+ = Eu 3+ > Gd 3+ > La 3+ > Zn 2+ = cation depleted. Except for Zn 2+, ranking for their activating potency of MLCK, the analogs can be arranged in a similar order.

The conformation of calmodulin: A substantial environmentally sensitive helical transition in Ca 4-calmodulin with potential mechanistic function

The conformation of Ca 4-calmodulin in solution, as assessed by far-UV peptide circular dichroism, contains significantly less α-helix than the proposed X-ray crystal structure. We now show that Ca 4-calmodulin adopts significant additional helical structure in solution in the presence of a helicogenic solvent (50%, v/v, aqueous 2,2,2-trifluoroethanol or 50%, v/v, methylpentane-5,5-diol). We suggest that the long continuous helix (residues 66–92 of the crystal structure)is not necessarily a normal feature of the calmodulin structure in solution, and may be due in part to the conditions of crystallisation. This result is supported by time-resolved tyrosine fluorescence anisotropy studies indicating that Ca 4-calmodulin in solution is an essentially compact globular structure which undergoes isotropic rotational motion. We conclude that, under appropriate ionic and apolar environmental conditions, Ca 4-calmodulin undergoes a substantial helical transition, which may involve residues in the central region of the molecule. Such a transition could have an important function in determining specificity and affinity in interactions of calmodulin with different target sequences of Ca 2+-dependent regulatory enzymes.

Separation of Tyrosine Phosphorylated Calmodulin from Calmodulin Using Non-Porous Anion-Exchange HPLC

Abstract Calmodulin is a protein which exerts control over various physiological processes by binding and modulating key enzymes after it undergoes a calcium-induced conformational change. The insulin receptor kinase has been shown to phosphorylate tyrosine 99, in the third calcium binding cleft of the protein. Since each molecule accommodates only four calcium ions, the introduction of phosphate into one of these calcium binding pockets may alter biological activity. To determine the effects of this modification, required the separation of phosphorylated calmodulin from the unphosphorylated form. This paper addresses the HPLC techniques we have used for the successful separation of various forms of calmodulin. Reversed-phase HPLC at pH 7.8 in the presence of 100μM CaCl2 or 1mM EGTA gave different elution profiles, reflecting the effect Ca++ has on calmodulin conformation. Such changes were not observed using 0.1% trifluoroacetic acid in mobile phases since calmodulin binds essentially no calcium at pH 2....

1H-NMR Studies of Calmodulin: The Modifying Effect of W-7 (N-(6-Aminohexyl)-5-Chloro-1-Naphthalenesulfonamide) on the Calcium-Induced Conformational Changes of Calmodulin

The effect of W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide), a calmodulin antagonist, on the calcium-bound conformation of calmodulin was studied by 1H-NMR at 400 MHz. W-7 affected the resonances of lle-27, Phe-68, Phe-92, lle-100, His-107 and Val-142. The resonances of Met-71, Met-72, Met-76, Phe-89 and Phe-141 may be affected by W-7. These findings suggest that W-7 binds to hydrophobic ammo acid residues, which almost occur in calcium-binding sites II, III and IV or their vicinity. The effect of W-7 on the structure of calmodulin was similar to that of other drugs, trifluoperazine, D600 and oxmetidme. Thus, those residues in the high-field methyl region, the methionine methyl region and the phenylalanine aromatic region of calmodulin, which were similarly affected by all four drugs, may be important at the interface for binding of calmodulin to the regulatory sites on target enzymes.

Antibody that recognizes conformations of calmodulin in the serum from patient with chronic active hepatitis

According to the effects of Ca++ on the reactivity, anti-calmodulin antibody in the sera of patients with autoimmune chronic active hepatitis was classified into three types, which were tentatively designated as type 1, 2 and 3. Type 1 antibody reacted with calmodulin only in the presence of free Ca++. Binding of type 2 antibody to calmodulin was inhibited by the presence of free Ca++. Type 3 antibody reacted with calmodulin regardless of the presence or absence of free Ca++. Thus the sera contained the populations of the antibody which recognized the different conformations of calmodulin molecule.

Peptide antisera as sequence-specific probes of protein conformational transitions: calmodulin exhibits calcium-dependent changes in antigenicity.

Local changes in conformation between the calcium-saturated and calcium-free forms of calmodulin were monitored using antisera to four peptides corresponding to three helical regions of the calcium-saturated protein. The N-terminal helix was monitored using antiserum to residues 9-19, calmodulin-(9-19); the C-terminal helix using antiserum to residues 141-148, calmodulin-(141-148); and the long central helix with antisera to residues 68-79 and 80-92, calmodulin-(68-79) and -(80-92). Crossreactivities of peptide antisera with calmodulin (either in the presence or absence of calcium) were determined using solution-phase and solid-phase immunoassays. When examined by the fluid-phase assay, all four peptides elicited antibody that precipitated radiolabeled apocalmodulin but not the calcium-saturated form of the protein. Similarly, when calmodulin was immobilized on a solid-support, only the calcium-free form readily bound the antibodies to calmodulin-(80-92) and -(141-148). In addition, the crossreactivity of antiserum to calmodulin-(68-79) with calcium-saturated calmodulin in solid phase was reduced by approximately equal to 40% relative to reactivity with apocalmodulin. According to the x-ray crystal structure of Ca2+-saturated calmodulin and the antigenic reactivity of calmodulin for the peptide antisera in the absence of calcium, the regions of the protein monitored by these antisera are exposed to the surface in both conformational states and probably accessible to specific antibodies. The apparent preference of peptide antibodies for one conformation of the molecule suggests that changes in the conformation of calmodulin occur in cognate sequences that are transformed by calcium from antigenic, flexible structures to less antigenic, relatively helical structures. Peptide antibodies may be employed as sequence-specific reporter molecules to monitor local conformational changes providing the cognate sequence is sterically accessible to antibody in both states but antigenic in only one.