Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Trad, Chafia H.; James, William L.; Bhardwaj, Anita; Butterfield, D. Allan

doi:10.1016/0165-022x(95)00016-9

Cited by 8 publications

(8 citation statements)

References 34 publications

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“…Earlier studies in our laboratory using other conditions of free radical‐induced oxidative stress, such as hydroxyl radical generation, 21 sepsis‐associated lipopolysaccharide, 22 or menadione 23 have shown W/S ratios to be lowered in each case. Hence, the W/S ratio can be used as a valuable marker of protein alterations.…”

Section: Markers Of Membrane Protein Damagementioning

confidence: 82%

“…24 Decreased W/S ratios indicate increased protein-protein interaction and decreased segmental motion and/or conformational changes in the proteins that were labeled; the converse is also the case. 24 Earlier studies in our laboratory using other conditions of free radical-induced oxidative stress, such as hydroxyl radical generation, 21 sepsis-associated lipopolysaccharide, 22 or menadione 23 have shown W/S ratios to be lowered in each case. Hence, the W/S ratio can be used as a valuable marker of protein alterations.…”

Section: Protein Conformational Changesmentioning

confidence: 95%

“…Several in vivo and in vitro models of oxidative stress have been developed and studied for this purpose. 12,21‐23 The focus of this review is a summary of work done in our laboratory on changes in protein structure and function in three in vivo models of free radical‐induced oxidative stress, namely, hyperoxia, ischemia‐reperfusion injury (IRI), and accelerated senescence, and the protection offered by the free radical scavenger, N‐tert ‐butyl‐α‐phenylnitrone (PBN), against these damages. This review also summarizes similar protein damage seen in an in vitro model of oxidative stress, that is, synaptosomes exposed to amyloid β‐peptide, a peptide implicated in the pathology of AD, and the protective effects of the antioxidant vitamin E against the ensuing damage.…”

Section: Oxidative Stress and Agingmentioning

confidence: 99%

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Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a

Butterfield

Koppal

Howard

et al. 1998

Annals of the New York Academy of Sciences

184

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The free radical theory of aging proposes that reactive oxygen species (ROS) cause oxidative damage over the lifetime of the subject. It is the cumulative and potentially increasing amount of accumulated damage that accounts for the dysfunctions and pathologies seen in normal aging. We have previously demonstrated that both normal rodent brain aging and normal human brain aging are associated with an increase in oxidative modification of proteins and in changes in plasma membrane lipids. Several lines of investigation indicate that one of the likely sources of ROS is the mitochondria. There is an increase in oxidative damage to the mitochondrial genome in aging and a decreased expression of mitochondrial mRNA in aging. We have used a multidisciplinary approach to the characterization of the changes that occur in aging and in the modeling of brain aging, both in vitro and in vivo. Exposure of rodents to acute normobaric hyperoxia for up to 24 h results in oxidative modifications in cytosolic proteins and loss of activity for the oxidation-sensitive enzymes glutamine synthetase and creatine kinase. Cytoskeletal protein spin labeling also reveals synaptosomal membrane protein oxidation following hyperoxia. These changes are similar to the changes seen in senescent brains, compared to young adult controls. The antioxidant spin-trapping compound N-tert-butyl-alpha-phenylnitrone (PBN) was effective in preventing all of these changes. In a related study, we characterized the changes in brain protein spin labeling and cytosolic enzyme activity in a series of phenotypically selected senescence-accelerated mice (SAMP), compared to a resistant line (SAMR1) that was derived from the same original parents. In general, the SAM mice demonstrated greater oxidative changes in brain proteins. In a sequel study, a group of mice from the SAMP8-sensitive line were compared to the SAMR1-resistant mice following 14 days of daily PBN treatment at a dose of 30 mg/kg. PBN treatment resulted in an improvement in the cytoskeletal protein labeling toward that of the normal control line (SAMR1). The results of these and related studies indicate that the changes in brain function seen in several different studies may be related to the progressive oxidation of critical brain proteins and lipids. These components may be critical targets for the beneficial effects of gerontotherapeutics both in normal aging and in disease of aging.

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Section: Markers Of Membrane Protein Damagementioning

confidence: 82%

Section: Protein Conformational Changesmentioning

confidence: 95%

Section: Oxidative Stress and Agingmentioning

confidence: 99%

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Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a

Butterfield

Koppal

Howard

et al. 1998

Annals of the New York Academy of Sciences

184

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“…Spin labeling is usually achieved post-translationally by in-vitro chemical reactions involving cysteines through disulfide formation [191, 192] or lysines [172]. …”

Section: Post-expression Labelingmentioning

confidence: 99%

Isotope Labeling for Solution and Solid-State NMR Spectroscopy of Membrane Proteins

Verardi

Traaseth

Masterson

et al. 2012

Advances in Experimental Medicine and Biology

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In this chapter, we summarize the isotopic labeling strategies used to obtain high-quality solution and solid-state NMR spectra of biological samples, with emphasis on integral membrane proteins (IMPs). While solution NMR is used to study IMPs under fast tumbling conditions, such as in the presence of detergent micelles or isotropic bicelles, solid-state NMR is used to study the structure and orientation of IMPs in lipid vesicles and bilayers. In spite of the tremendous progress in biomolecular NMR spectroscopy, the homogeneity and overall quality of the sample is still a substantial obstacle to overcome. Isotopic labeling is a major avenue to simplify overlapped spectra by either diluting the NMR active nuclei or allowing the resonances to be separated in multiple dimensions. In the following we will discuss isotopic labeling approaches that have been successfully used in the study of IMPs by solution and solid-state NMR spectroscopy.

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“…A cysteine side chain is introduced into the protein structure with site‐directed mutagenesis and is subsequently chemically modified with a nitroxide radical. The most commonly used nitroxide radical is the methanethiosulfonate spin‐label (MTSSL), which, upon attachment to a cysteine, yields the spin‐labeled side chain, commonly termed R1 [16,17]. The dynamical modes of the nitroxide are a combination of rotary diffusion of the macromolecule, internal bond isomerization of the spin‐label and backbone fluctuations.…”

mentioning

confidence: 99%

Structural features and dynamics of a cold‐adapted alkaline phosphatase studied by EPR spectroscopy

Heidarsson¹,

Sigurdsson²,

Ásgeirsson³

2009

The FEBS Journal

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EPR spectroscopy, performed after site‐directed spin‐labeling, was used to study structural dynamics in a cold‐adapted alkaline phosphatase (EC 3.1.1.1). Differences in the structural environment of six spin‐labeled side chains allowed them to be classified (with reference to previously obtained mobility maps) as belonging to loop positions (either relatively surface exposed or in structural contact) or helix positions (surface exposed, in contact, or buried). The mobility map constructed in the present study provides structural information that is in broad agreement with the location in the crystal structure. All but one of the chosen serine‐to‐cysteine mutations reduced activity considerably and this coincided with improved thermal stability. The effect of spin‐labeling on enzyme function ranged from nonperturbing to an almost complete loss of activity. In the latter case, treatment with a thiol reagent reactivated the enzyme, indicating relief of steric hindrance to the catalytic process. Two mutations of an active‐site residue W274 (K328 in Escherichia coli alkaline phosphatase), known to reduce activity and increase stability of Vibrio alkaline phosphatase, gave a coincidental reduction in mobility of a nearby spin‐label located at C67, as determined by EPR spectroscopy. This suggests that movement of the helix carrying C67 and the closely positioned nucleophilic S65 is interconnected with catalytic events.

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Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Cited by 8 publications

References 34 publications

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a

Isotope Labeling for Solution and Solid-State NMR Spectroscopy of Membrane Proteins

Structural features and dynamics of a cold‐adapted alkaline phosphatase studied by EPR spectroscopy

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Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Cited by 8 publications

References 34 publications

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin Ea

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin Ea

Isotope Labeling for Solution and Solid-State NMR Spectroscopy of Membrane Proteins

Structural features and dynamics of a cold‐adapted alkaline phosphatase studied by EPR spectroscopy

Contact Info

Product

Resources

About

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a

Structural and Functional Changes in Proteins Induced by Free Radical‐mediated Oxidative Stress and Protective Action of the Antioxidants N‐tert‐Butyl‐α‐phenylnitrone and Vitamin E^a