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

Strasburg, Gale M.; Hogan, Margaret E.; Birmachu, Woubalem; Thomas, David D.; Louis, Charles F.

doi:10.1016/s0021-9258(19)57426-9

Cited by 78 publications

(60 citation statements)

References 34 publications

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“…These experiments were facilitated by the use of spinach CaM which contains a single cysteine residue that can be readily covalently labeled with reporter molecules (Toda et al, 1985;Mills et al, 1988). Strasburg et al (1988) have demonstrated that this cysteine residue can be specifically labeled with 1,5-IAEDANS in the presence of 6 N guanidine. Furthermore, the position of this cysteine, residue 26 of spinach CaM (Lukas et al, 1984;Toda et al, 1988), is conveniently located near the extreme amino-terminal end of the three-dimensional structure of the molecule (Babu et al, 1985(Babu et al, ,1988; Figure 3).…”

Section: Resultsmentioning

confidence: 99%

Fluorescence energy transfer analysis of calmodulin.cntdot.peptide complexes

Chapman¹,

Alexander²,

Vorherr³

et al. 1992

Biochemistry

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The interactions between calmodulin and the tryptophan residues of synthetic peptides corresponding to the calmodulin binding domains of skeletal muscle myosin light-chain kinase and the plasma membrane calcium pump were examined. The single tryptophan residue contained in each peptide became relatively immobilized and inaccessible to iodide ion upon binding to calmodulin, indicating that the indole side chain was inserted into a hydrophobic cleft in the surface of calmodulin. Fluorescence energy transfer from peptidyl tryptophan residues to an AEDANS moiety attached to cysteine-26 of spinach calmodulin was measured. Included in these analyses was a tryptophan-containing peptide analog of the calmodulin binding domain of neuromodulin. These data indicated that the indole ring of each peptide inserted 32-35 A away from cysteine-26 and may therefore interact with the carboxyl-terminal lobe of CaM in its "bent" conformation [Persechini & Kretsinger (1988a) J. Cardiovasc. Pharmacol. 12 (Suppl 5), S1-S12; Ikura et al. (1992) Science 256, 632-638; Vorherr et al. (1992) Eur. J. Biochem. 204, 931-937]. The interchange of tryptophan-3 and phenylalanine-21 of the calcium pump peptide increased the efficiency of energy transfer to the AEDANS-moiety approximately 12-fold, reducing the calculated distance to 20 A. These data suggest that phenylalanine-21 of the calcium pump peptide interacts with the hydrophobic cleft in the amino-terminal lobe of CaM.

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Section: Resultsmentioning

confidence: 99%

Fluorescence energy transfer analysis of calmodulin.cntdot.peptide complexes

Chapman¹,

Alexander²,

Vorherr³

et al. 1992

Biochemistry

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“…CaM was purified from wheat germ according to the modified procedure (Strasburg et al, 1988) of Yoshida et al (1983). Labeling of purified wheat germ CaM was carried out by first reducing the protein with 0.1 M dithiothreitol in 6 M guanidine hydrochloride, 25 mM Hepes (pH 7.5), and 1 mM EDTA.…”

Section: Methodsmentioning

confidence: 99%

pH-dependent conformational changes of wheat germ calmodulin

Wang¹

1989

Biochemistry

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Fluorescence energy transfer measurements were carried out between landmarks on wheat germ calmodulin to measure the interdomain distance. Tb3+ ions bound at the four Ca2+-binding sites were used as energy donors, and an organic chromophore, [4-(dimethylamino)-phenyl-4'-azophenyl]maleimide, attached to the single cysteine residue at position 27, was used as the acceptor. At pH's near neutrality all bound Tb3+ ions emit luminescence with shortened lifetimes as a result of energy transfer to the acceptor; at pH 5, however, part of the metal emission becomes unquenched. When the protein is subjected to limited digestion with trypsin in the presence of Ca2+, resulting in the formation of two fragments, each corresponding to half of the molecule, the decay of Tb3+ emission is no longer pH sensitive. These results suggest that, like rabbit skeletal troponin C [Wang, C.-L. A., Zhan, Q., Tao, T., & Gergely, J. (1987) J. Biol. Chem. 257, 8372-8375], wheat germ calmodulin exists in a relatively compact conformation at neutral pH's, but becomes more elongated at pH 5.

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“…CaM and bovine 20S proteasome were purified essentially as described previously (18)(19)(20)(21). Briefly, after induction of reticulocytosis, washed red blood cells (.90% reticulocytes) were collected and lysed in 1 mM DTT.…”

Section: Protein Purificationmentioning

confidence: 99%

Tertiary Structural Rearrangements upon Oxidation of Methionine145 in Calmodulin Promotes Targeted Proteasomal Degradation

Sacksteder

Whittier

Xiong

et al. 2006

Biophysical Journal

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The selectivity underlying the recognition of oxidized calmodulin (CaM) by the 20S proteasome in complex with Hsp90 was identified using mass spectrometry. We find that degradation of oxidized CaM (CaMox) occurs in a multistep process, which involves an initial cleavage that releases a large N-terminal fragment (A1-F92) as well as multiple smaller carboxyl-terminus peptides ranging from 17 to 26 amino acids in length. These latter small peptides are enriched in methionine sulfoxides (MetO), suggesting a preferential degradation around MetO within the carboxyl-terminal domain. To confirm the specificity of CaMox degradation and to identify the structural signals underlying the preferential recognition and degradation by the proteasome/Hsp90, we have investigated how the oxidation of individual methionines affect the degradation of CaM using mutants in which all but selected methionines in CaM were substituted with leucines. Substitution of all methionines with leucines except Met144 and Met145 has no detectable effect on the structure of CaM, permitting a determination of how site-specific substitutions and the oxidation of Met144 and Met145 affects the recognition and degradation of CaM by the proteasome/Hsp90. Comparable rates of degradation are observed upon the selective oxidation of Met144 and Met145 in CaM-L7 relative to that observed upon oxidation of all nine methionines in wild-type CaM. Substitution of leucines for either Met144 or Met145 promotes a limited recognition and degradation by the proteasome that correlates with decreases in the helical content of CaM. The specific oxidation of Met144 has little effect on rates of proteolytic degradation by the proteasome/Hsp90 or the structure of CaM. In contrast, the specific oxidation of Met145 results in both large increases in the rate of degradation by the proteasome/Hsp90 and significant circular dichroic spectral shape changes that are indicative of changes in tertiary rather than secondary structure. Thus, tertiary structural changes resulting from the site-specific oxidation of a single methionine (i.e., Met145) promote the degradation of CaM by the proteasome/Hsp90, suggesting a mechanism to regulate cellular metabolism through the targeted modulation of CaM abundance in response to oxidative stress.

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

Cited by 78 publications

References 34 publications

Fluorescence energy transfer analysis of calmodulin.cntdot.peptide complexes

Fluorescence energy transfer analysis of calmodulin.cntdot.peptide complexes

pH-dependent conformational changes of wheat germ calmodulin

Tertiary Structural Rearrangements upon Oxidation of Methionine145 in Calmodulin Promotes Targeted Proteasomal Degradation

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