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Title: Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation

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Publication Date:
Research Org.:
Environmental Molecular Sciences Laboratory (EMSL)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
OSTI Identifier:
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; Journal Volume: 45; Journal Issue: 15
Country of Publication:
United States

Citation Formats

Yan, Ping, Xiong, Yijia, Chen, Baowei, Negash, Sewite, Squier, Thomas C., and Mayer, M. Uljana. Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation. United States: N. p., 2006. Web. doi:10.1021/bi052395a.
Yan, Ping, Xiong, Yijia, Chen, Baowei, Negash, Sewite, Squier, Thomas C., & Mayer, M. Uljana. Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation. United States. doi:10.1021/bi052395a.
Yan, Ping, Xiong, Yijia, Chen, Baowei, Negash, Sewite, Squier, Thomas C., and Mayer, M. Uljana. Sat . "Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation". United States. doi:10.1021/bi052395a.
title = {Fluorophore-Assisted Light Inactivation of Calmodulin Involves Singlet-Oxygen Mediated Cross-Linking and Methionine Oxidation},
author = {Yan, Ping and Xiong, Yijia and Chen, Baowei and Negash, Sewite and Squier, Thomas C. and Mayer, M. Uljana},
abstractNote = {},
doi = {10.1021/bi052395a},
journal = {Biochemistry},
number = 15,
volume = 45,
place = {United States},
year = {Sat Apr 01 00:00:00 EST 2006},
month = {Sat Apr 01 00:00:00 EST 2006}
  • Fluorophore-assisted light inactivation (FALI) can permit the targeted inactivation of tagged proteins and, when used with cell-permeable multiuse affinity probes (MAPs), offers important advantages in identifying physiological function, because targeted protein inactivation is possible with spatial and temporal control. However, reliable applications of FALI, also known as chromophore-assisted light inactivation (CALI) with fluorescein derivatives, have been limited by lack of mechanistic information regarding sensitive proteins. To permit the rational inactivation of targeted proteins we have identified the oxidizing species and the susceptibility of specific amino acids to modification using the calcium regulatory protein calmodulin (CaM) that, like many essential proteins,more » regulates signal transduction through the reversible association with a large number of target proteins. Following the covalent and rigid attachment of 4’,5’-bis (1,3,2-dithoarsolan-2-yl) fluorescein (FlAsH) to helix A, we have identified light-dependent oxidative modifications of endogenous methionines to their corresponding methionine sulfoxides. Initial rates of methionine oxidation correlate with surface accessibility and are insensitive to the distance between the bound fluorophore and individual methionines, which vary between ~7 and 40 Å. In addition, we observed a rapid reaction of histidines, which results in selective cross-linking with binding partners corresponding to the CaM-binding sites of smooth myosin light chain kinase and ryanodine receptor. Our results provide a rationale for proteomic screens using FALI to inhibit the function of many signaling proteins, which, like CaM, commonly present methionines at binding interfaces. Likewise, properly placed histidines will permit the capture and identification of binding partners associated with protein complexes.« less
  • The C-terminus of calmodulin (CaM) functions as a sensor of oxidative stress, with oxidation of methionine 144 and 145 inducing a nonproductive association of the oxidized CaM with the plasma membrane Ca 2+-ATPase (PMCA) and other target proteins to downregulate cellular metabolism. To better understand the structural underpinnings and mechanism of this switch, we have engineered a CaM mutant (CaM-L7) that permits the site-specific oxidation of M144 and M145, and we have used NMR spectroscopy to identify structural changes in CaM and CaM-L7 and changes in the interactions between CaM-L7 and the CaM-binding sequence of the PMCA (C28W) due tomore » methionine oxidation. In CaM and CaM-L7, methionine oxidation results in nominal secondary structural changes, but chemical shift changes and line broadening in NMR spectra indicate significant tertiary structural changes. For CaM-L7 bound to C28W, main chain and side chain chemical shift perturbations indicate that oxidation of M144 and M145 leads to large tertiary structural changes in the C-terminal hydrophobic pocket involving residues that comprise the interface with C28W. Smaller changes in the N-terminal domain also involving residues that interact with C28W are observed, as are changes in the central linker region. At the C-terminal helix, 1H α, 13C α, and 13CO chemical shift changes indicate decreased helical character, with a complete loss of helicity for M144 and M145. Using 13C-filtered, 13C-edited NMR experiments, dramatic changes in intermolecular contacts between residues in the C-terminal domain of CaM-L7 and C28W accompany oxidation of M144 and M145, with an essentially complete loss of contacts between C28W and M144 and M145. We propose that the inability of CaM to fully activate the PMCA after methionine oxidation originates in a reduced helical propensity for M144 and M145, and results primarily from a global rearrangement of the tertiary structure of the C-terminal globular domain that substantially alters the interaction of this domain with the PMCA.« less
  • The one-electron (1e) oxidation of organic sulfides and methionine (Met) constitutes an important reaction mechanism in vivo.1,2 Evidence for a Cu(II)-catalyzed oxidation of Met35 in the Alzheimer's disease -amyloid peptide was obtained,3 and, based on theoretical studies, Met radical cations were proposed as intermediates.4 In the structure of -amyloid peptide, the formation of Met radical cations appears to be facilitated by a preexisting close sulfur-oxygen (S-O) interaction between the Met35 sulfur and the carbonyl oxygen of the peptide bond C-terminal to Ile31.5 Substitution of Ile31 with Pro31 abolishes this S-O interaction,5 significantly reducing the ability of -amyloid to reduce Cu(II),more » and converts the neurotoxic wild-type -amyloid into a non-toxic peptide.6 The preexisting S-O bond characterized for wild-type -amyloid suggests that electron transfer from Met35 to Cu(II) is supported through stabilization of the Met radical cation by the electron-rich carbonyl oxygen, generating an SO-bonded7 sulfide radical cation (Scheme 1, reaction 1).5« less
  • We have developed a method for rapidly quantifying the extent to which the functionally important Met144 and Met145 residues near the C-terminus of calmodulin (CaM) are converted to the corresponding sulfoxides, Met(O). The method utilizes a whole protein collision induced dissociation (CID) approach on an electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer. Using standards of CaM oxidized by hydrogen peroxide (H2O2) or peroxynitrite (ONOO-), we demonstrated that CID fragmentation of the protein ions resulted in a series of C-terminal singly charged y1?y15 ions. Fragments larger than y4 exhibited mass shifts of +16 or +32 Da, corresponding to oxidation of onemore » or two methionines, respectively. To assess the extent of oxidative modification for Met144 and Met145 to Met(O), we averaged the ratio of intensities for yn, yn +16, and yn +32 ions, where n = 6?9. By alternating MS and CID scans at low and high collision energies, this technique allowed us to rapidly determine both the distribution of intact CaM oxiforms and the extent of oxidative modification in the C-terminal region of the protein in a single run. We have applied the method to studies of the repair of fully oxidized CaM by methionine sulfoxide reductases (MsrA and MsrB), which normally function in concert to reduce the S and R stereoisomers of methionine sulfoxide. We found that repair of Met(O)144 and Met(O)145 did not go to completion, but was more efficient than average Met repair. Absence of complete repair is consistent with previous studies showing that accumulation of methionine sulfoxide in CaM can occur during aging.« less
  • Highlights: • We measured the distance distribution between two spin labels on calmodulin by DEER. • Two structural states, open and closed, were resolved at both low and high Ca. • Ca shifted the equilibrium toward the open state by a factor of 13. • Methionine oxidation, simulated by glutamine substitution, decreased the Ca effect. • These results have important implications for aging in muscle and other tissues. - Abstract: We have used electron paramagnetic resonance (EPR) to examine the structural impact of oxidizing specific methionine (M) side chains in calmodulin (CaM). It has been shown that oxidation of eithermore » M109 or M124 in CaM diminishes CaM regulation of the muscle calcium release channel, the ryanodine receptor (RyR), and that mutation of M to Q (glutamine) in either case produces functional effects identical to those of oxidation. Here we have used site-directed spin labeling and double electron–electron resonance (DEER), a pulsed EPR technique that measures distances between spin labels, to characterize the structural changes resulting from these mutations. Spin labels were attached to a pair of introduced cysteine residues, one in the C-lobe (T117C) and one in the N-lobe (T34C) of CaM, and DEER was used to determine the distribution of interspin distances. Ca binding induced a large increase in the mean distance, in concert with previous X-ray crystallography and NMR data, showing a closed structure in the absence of Ca and an open structure in the presence of Ca. DEER revealed additional information about CaM’s structural heterogeneity in solution: in both the presence and absence of Ca, CaM populates both structural states, one with probes separated by ∼4 nm (closed) and another at ∼6 nm (open). Ca shifts the structural equilibrium constant toward the open state by a factor of 13. DEER reveals the distribution of interprobe distances, showing that each of these states is itself partially disordered, with the width of each population ranging from 1 to 3 nm. Both mutations (M109Q and M124Q) decrease the effect of Ca on the structure of CaM, primarily by decreasing the closed-to-open equilibrium constant in the presence of Ca. We propose that Met oxidation alters CaM’s functional interaction with its target proteins by perturbing this Ca-dependent structural shift.« less