Differential trace labeling of calmodulin: Investigation of binding sites and conformational states by individual lysine reactivities. Effects of β-endorphin, trifluoperazine, and ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (original) (raw)
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Calcium effects on calmodulin lysine reactivities
Archives of Biochemistry and Biophysics, 1987
The differential reactivities of individual lysines on porcine testicular calmodulin were determined by trace labeling with high specific activity [3H]acetic anhydride as a function of the molar ratio of Ca2+ to calmodulin. In progressing from the Ca2+-depleted form of the protein to a Ca2+:calmodulin molar ratio of 51, six of the seven lysyl residues exhibited a modest 1.5 to 3.0-fold increase in reactivity. Lys 75, in contrast, was enhanced in reactivity greater than 20-fold. When the change in reactivity of each lysine was normalized as a percentage of the maximum change, most of the residues were found to fall into two distinct classes. One class, comprising lysines 94 and 148 from the two carboxy terminal Ca2+-binding domains 3 and 4, respectively, exhibited about 90% of their reactivity change when the Ca2+:calmodulin molar ratio was 2:1, and these residues were perturbed very little upon further addition of Ca '+ The other class, encompassing . lysines 13,21, and 30 from the amino terminal domain 1 and Lys 75 from the extended helix connecting the two globular lobes of calmodulin, underwent most of their overall reactivity change (55-'70%) between 2 and 5 equivalents of Ca2+ per mol of calmodulin. Lys 77 was distinct in its pattern of change, undergoing approximately equal changes with each Ca2+ increment. These results are consistent with a model where Ca2+ first binds to the two carboxy terminal sites of calmodulin with no apparent preference, concomitant with minor alterations in the microenvironments of lysines in the unoccupied amino terminal domains. The third and fourth Ca2+ ions then bind to these latter two domains, again with no evidence of preference, with little change in the lysine reactivities at the carboxy terminus of the molecule. The environments of groups in the central helix appear to undergo changes in a manner that reflects their proximity to the amino and carboxy terminal domains. In the course of this work, it was found that Lys 94 in apocalmodulin is specifically perturbed by the addition of EGTA, suggesting that the chelating agent may interact with calmodulin at or near the third Ca2+-binding domain. 0
Effects of interaction with calcineurin on the reactivities of calmodulin lysines
The Journal of biological chemistry, 1988
Calmodulin was trace labeled by acetylation with [3H]acetic anhydride in the presence and absence of a 30% molar excess of the phosphatase calcineurin; phenylalanine was included in the reaction mixtures as an internal standard. The level of 3H acetylation of each of the 7 lysines was determined and corrected for differences arising from reaction conditions using the labeling of the internal standard, following procedures that are closely similar to those used in a previous study of the interaction of calmodulin with myosin light chain kinase (Jackson, A. E., Carraway, K. L., III, Puett, D., and Brew, K. (1986) J. Biol. Chem. 261, 12226-12232). The interaction with calcineurin was found to produce a 10-fold reduction in the acetylation of lysine 75, with lesser but significant effects on lysines 21 and 148. A small but reproducible perturbation of lysine 77 was also observed. The results are similar to those that are produced by the interaction with myosin light chain kinase. Howeve...
The effects of deletions in the central helix of calmodulin on enzyme activation and peptide binding
The Journal of Biological Chemistry, 1989
Using site-directed mutagenesis we have expressed in Escherichia coli three engineered calmodulins (CaM) containing deletions in the solvent-exposed region of the central helix. These are CaMA84, Glu-84 removed; CaMA83-84, Glu-83 and Glu-84 removed; and CaMA81-84, Ser-81 through Glu-84 removed. The abilities of these proteins to activate skeletal muscle myosin light chain kinase, plant NAD kinase, and bovine brain calcineurin activities were determined, as were their abilities to bind a synthetic peptide based on the calmodulin-binding domain of skeletal muscle myosin light chain kinase. Similar results were obtained with all three deletion proteins. V , values for enzymes activated by the deletion proteins are all within 10-20% of those values obtained with bacterial control calmodulin. Relative to bacterial control values, changes in K,,, or K,j values associated with the deletions are all less than an order of magnitude: KaCt values for NAD kinase and myosin light chain kinase are increased 5-7-fold, K d values for binding of the synthetic peptide are increased 4-7-fold, and K,,, values for calcineurin are increased only 1-%fold. In assays of NAD kinase and myosin light chain kinase activation some differences between bovine calmodulin and bacterial control calmodulin were observed. With NAD kinase, K,,, values for the bacterial control protein are increased 4-fold relative to values for bovine calmodulin, and V , values are increased by 50%; with myosin light chain kinase, K,,, values are increased 2fold and V , values are decreased 10-15'70 relative to those values obtained with bovine calmodulin. These differences between bacterial control and bovine calmodulins probably can be attributed to known differences in postranslational processing of calmodulin in bacterial and eucaryotic cells. No differences between bovine and control calmodulins were observed in assays of calcineurin activation or peptide binding. Our observations indicate that contacts with the deleted The nucleotide sequence(s) reported in thispaper has been submitted 504 729.
Biochemical Journal, 1992
An indirect enzyme-linked immunosorbent assay has been used to study the interactions between calmodulin and two calmodulin antagonists, trifluoperazine and a neuropeptide isolated from the hypothalamus. The binding of a monospecific anti-calmodulin antibody, raised in rabbit against dinitrophenylated calmodulin, to calmodulin was tested at various concentrations of these drugs under equilibrium conditions. Trifluoperazine at low concentrations stimulated, but at relatively high concentrations inhibited, immunocomplex formation. The neuropeptide displaced the antibody from calmodulin at nanomolar concentrations. Enzyme-linked immunosorbent assays were also carried out with the large tryptic fragments of calmodulin. The results suggest that (i) the C-terminal fragment binds the antibody with an affinity which is comparable with that of intact calmodulin; (ii) the neuropeptide can form complexes with both N-and C-terminal fragments, but with two orders of magnitude less activity in case of the C-terminal fragment; and (iii) trifluoperazine does not stimulate antibody binding to the C-terminal fragment. Therefore the tertiary structure of calmodulin must be intact to ensure long-distance interactions between the binding sites of trifluoperazine, the neuropeptide and the antibody. These interactions may produce distinct conformers of calmodulin which may exhibit altered potency, not only for antibody binding but also for stimulation/inhibition of target enzymes.
Journal of Molecular Recognition, 1995
Calmodulin, similarly to many other Ca"-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, CAMl, CAM2 and CAM4) were purified and characterized. MAb CAMl was identified as an IgGl while mAbs CAM2 and CAM4 belong to IgM class. Additivity ELISA showed that mAb CAMl binds to an epitope located remote from the epitopes recognized by the other two mAbs, while mAbs CAM2 and CAM4 recognize close epitopes. MAb CAMl was found to be especially sensitive to the conformational state of calmodulin in the presence of CaZ+ ions. The interactions of mAbs CAM2 and CAM4 with calmodulin are only slightly affected by Ca" removal. In addition mAb CAMl failed to recognize other calmodulin molecules, such as spinach and various plant recombinant calmodulins, while mAbs CAM2 and CAM4 share common epitopes with the above molecules
On the mechanism of interaction between calmodulin and calmodulin-dependent proteins
Biochemistry and Cell Biology, 1983
- On the mechanism of interaction between calmodulin and calmodulin-dependent proteins. Can. J. Biochem. Cell Biol. 61. 91 1-920 Molecular and kinetic studies of the interaction between calmodulin and calmodulin-dependent proteins have been reviewed. Several calmodulin-dependent proteins have been purified to homogeneity and characterized in terms of subunit structure in recent years. The results indicate that these proteins do not contain a common subunit as the basis of calmodulin binding. A monoclonal antibody capable of interacting with several calmodulin-dependent proteins has been obtained, suggesting that these proteins contain common structure. It seems that hybridoma technology may be used for probing calmodulin-binding domains in the calmodulin-dependent proteins. Using a fluorescent-labelled cyclic nucleotide phosphodiesterase, the interaction between calmodulin and the enzyme in the absence of ~a " can be demonstrated, and the equilibrium constant of the reaction can be determined. The study further defines the multiple interactions in the activation of the cyclic nucleotide phosphodiesterase by ~a " and calmodulin. Previous kinetic results along with the present results are summarized and used to elucidate the regulatory significance of the multiple ca2+-binding of calmodulin. 1983) On the mechanism of interaction between calmodulin and calmodulin-dependent proteins. Can. J. Biochem. Cell Biol. 61, 9 1 1-920 Nous rCvisons les Ctudes molCculaires et cinktiques de l'interaction entre la calmoduline et les protCines ddpandantes de la calmoduline. Au cours des derni&res annkes, plusieurs protkines ddpendantes de la calmoduline ont Ct C purifikes jusqu'h homogCnCitC et caractCrisCes en temes de structure sous-unitaire. Les rksultats montrent que ces protCines ne contiennent pas de sous-unit6 commune qui serait a la base de la liaison h la calmoduline. On a obtenu un anticorps monoclonal capable de rdagir avec plusieurs protCines ddpndantes de la calmoduline; c'est 18 un indice que ces protCines contiennent une structure commune.
Biochemistry, 1991
Peptide-induced conformational changes in five isofunctional mutants of calmodulin (CaM), each bearing a single tryptophan residue either at the seventh position of each of the four calcium-binding loops (Le., amino acids 26, 62, 99, and 135) or in the central helix (amino acid 81) were studied by using fluorescence spectroscopy. The peptides RS20F and RS20CK correspond to CaM-binding amino acid sequence segments of either nonmuscle myosin light chain kinase (nmMLCK) or calmodulin-dependent protein kinase I1 (CaMPK-11), respectively. Both steady-state and time-resolved fluorescence data were collected from the various peptide-CaM complexes. Steady-state fluorescence intensity measurements indicated that, in the presence of an excess of calcium, both peptides bind to the calmodulin mutants with a 1:l stoichiometry. The tryptophans located in loops I and IV exhibited red-shifted emission maxima (356 nm), high quantum yields (0.3), and long average lifetimes (6 ns). They responded in a similar manner to peptide binding, by only slight changes in their fluorescence features. In contrast, the fluorescence intensity of the tryptophans in loops I1 and I11 decreased markedly, and their fluorescence spectrum was blue-shifted upon peptide binding. Analysis of the tryptophan fluorescence decay of the last mentioned calmodulins supports a model in which the equilibrium between two (Trp-99) or three (Trp-62) states of these tryptophan residues, each characterized by a different lifetime, was altered toward the blue-shifted short lifetime component upon peptide binding. Taken together, these data provide new evidence that both lobes of calmodulin are involved in peptide binding. Both peptides induced similar changes in the fluorescence properties of the tryptophan residues located in the calcium-binding loops, with the exception of calmodulin with Trp-135. For this last mentioned calmodulin, slight differences were observed. Tryptophan in the central helix responded differently to RS2OF and RS20CK binding. RS20F binding induced a red-shift in the emission maximum of Trp-8 1 while RS20CK induced a blue-shift. The quenching rate of Trp-8 1 by iodide was slightly reduced upon RS20CK binding, while RS20F induced a 2-fold increase. These results provide evidence that the environment of Trp-81 is different in each case and are, therefore, consistent with the hypothesis that the central helix can play a differential role in the recognition of, or response to, CaM-binding structures. Calmodulin (CaM)' is a ubiquitous protein involved in the regulation of a wide variety of calcium-dependent intracellular processes as varied as motivity, secretion, glycogen metabolism, synaptic transmission, transport of anions, and cyclic nucleotide metabolism [for reviews, see Rasmussen et al. (1984) and Manalan and Klee (1 984)]. Upon calcium binding, calmodulin apparently undergoes conformational changes, which enable it to bind to and activate target enzymes such as calmodulin-stimulated phosphodiesterase, plasma membrane Ca2+ pump, myosin light chain kinase, and calmodulin-dependent protein kinase 11 (Cox et al., 1984; Stoclet et al., 1987). The interaction of calmodulin with its target enzymes is very little known at the molecular level [for a review, see Blumenthal and Krebs (1 988)]. It is thought that most calmodulin-dependent enzymes contain specific regions that recognize and bind to calmodulin with high affinity. Such putative calmodulin-binding sites have been found in, e.g., skeletal muscle
Intra- and Interdomain Effects Due to Mutation of Calcium-binding Sites in Calmodulin
Journal of Biological Chemistry, 2010
The IQ-motif protein PEP-19, binds to the C-domain of calmodulin (CaM) with significantly different k on and k off rates in the presence and absence of Ca 2؉ , which could play a role in defining the levels of free CaM during Ca 2؉ transients. The initial goal of the current study was to determine whether Ca 2؉ binding to sites III or IV in the C-domain of CaM was responsible for affecting the kinetics of binding PEP-19. EF-hand Ca 2؉binding sites were selectively inactivated by the common strategy of changing Asp to Ala at the X-coordination position. Although Ca 2؉ binding to both sites III and IV appeared necessary for native-like interactions with PEP-19, the data also indicated that the mutations caused undesirable structural alterations as evidenced by significant changes in amide chemical shifts for apoCaM. Mutations in the C-domain also affected chemical shifts in the unmodified N-domain, and altered the Ca 2؉ binding properties of the N-domain. Conversion of Asp 93 to Ala caused the greatest structural perturbations, possibly due to the loss of stabilizing hydrogen bonds between the side chain of Asp 93 and backbone amides in apo loop III. Thus, although these mutations inhibit binding of Ca 2؉ , the mutated CaM may not be able to support potentially important native-like activity of the apoprotein. This should be taken into account when designing CaM mutants for expression in cell culture.