Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane

Bayley, Hagan; Knowles, Jeremy R.

doi:10.1021/bi00558a001

Cited by 91 publications

(45 citation statements)

References 51 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…First, INA is an inert compound under normal conditions and is activated only when excited by far-UV light [18]. Second, INA partitions rapidly into lipid bilayers, with a partition coefficient of 163,000 [35], and is virtually absent in aqueous medium. In the lipid bilayer, INA can be triggered by UV irradiation, to alkylate the acceptor moieties present therein.…”

Section: Discussionmentioning

confidence: 99%

Ebola Virus Inactivation with Preservation of Antigenic and Structural Integrity by a Photoinducible Alkylating Agent

et al. 2007

View full text Add to dashboard Cite

Current methods for inactivating filoviruses are limited to high doses of irradiation or formalin treatment, which may cause structural perturbations that are reflected by poor immunogenicity. In this report, we describe a novel inactivation technique for Zaire Ebola virus (ZEBOV) that uses the photoinduced alkylating probe 1,5-iodonaphthylazide (INA). INA is incorporated into lipid bilayers and, when activated by ultraviolet irradiation, alkylates the proteins therein. INA treatment of ZEBOV resulted in the complete loss of infectivity in cells. Results of electron microscopy and virus-capture assays suggested the preservation of conformational surface epitopes. Challenge with 50,000 pfu of INA-inactivated, mouse-adapted ZEBOV did not cause disease or death in mice. A single vaccination with INA-inactivated ZEBOV (equivalent to 5 x 10(4) pfu) protected mice against lethal challenge with 1000 pfu of ZEBOV. INA-inactivated virus induced a protective response in 100% of mice when administered 3 days before challenge. Thus, INA may have significant potential for the development of vaccines and immunotherapeutics for filoviruses and other enveloped viruses.

show abstract

Section: Discussionmentioning

confidence: 99%

Ebola Virus Inactivation with Preservation of Antigenic and Structural Integrity by a Photoinducible Alkylating Agent

et al. 2007

View full text Add to dashboard Cite

show abstract

“…Various reagents of this type have been developed and their properties have been reviewed (3-7). 5-Iodonaphthalene-1-azide (Ina) was used frequently and proved to be efficient in labeling hydrophobic domains of membrane proteins (8)(9)(10)(11)(12).…”

mentioning

confidence: 99%

“…Ina is highly hydrophobic (partition coefficient >105) (11) and partitions efficiently into the lipid bilayer of various cellular membranes. Upon photolysis (Xmax = 310 nm, E -21,400 M-1 cm-1) with the 314-nm line of a mercury lamp, it is rapidly and efficiently converted into the reactive nitrene, which inserts covalently into intrinsic membrane proteins that contain lipid-embedded domains and, to a lesser extent, into phospholipids.…”

mentioning

confidence: 99%

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

Raviv¹,

Salomon²,

Gitler³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

The apolar azide of 5-iodonaphthalene-1-azide (Ina) partitions into the lipid bilayer of biological membranes. Upon photolysis at 314 nm, it is rapidly converted into the reactive nitrene, which efficiently attaches covalently to lipid-embedded domains of proteins and, to a lesser extent, to membrane phospholipids. Above 370 nm, Ina absorption is negligible and photolysis at these wavelengths does not occur. However, on addition of the photosensitizing molecule 3-aminopyrene, trifluoperazine, or 8-anilinonaphthalene-1-sulfonate, followed by irradiation at 380 nm, efficient conversion of Ina to reactive species was observed, as measured by ['251]Ina-labeling of membrane proteins and inactivation of the hormonal response of adenylate cyclase. Irradiation at 480 nm in the presence of a fluorescein derivative of n-undecylamine also resulted in a pattern of [125] Klip and Gitler (1, 2) introduced the use of highly lipid-soluble azides for the labeling of proteins in membranes. Various reagents of this type have been developed and their properties have been reviewed (3-7). 5-Iodonaphthalene-1-azide (Ina) was used frequently and proved to be efficient in labeling hydrophobic domains of membrane proteins (8-12).Ina is highly hydrophobic (partition coefficient >105) (11) and partitions efficiently into the lipid bilayer of various cellular membranes. Upon photolysis (Xmax = 310 nm, E -21,400M-1 cm-1) with the 314-nm line of a mercury lamp, it is rapidly and efficiently converted into the reactive nitrene, which inserts covalently into intrinsic membrane proteins that contain lipid-embedded domains and, to a lesser extent, into phospholipids. When photoactivated in the plasma membrane, Ina blocks transduction of hormonal stimulation of adenylate cyclase by uncoupling of the hormone receptor from the enzyme (13).It was of interest to determine whether energy-transfer processes could be used to photoactivate a discrete fraction of the azide molecules present in a membrane or cell.Resonance energy transfer could not be used because Ina absorbs light down to 260 nm and any donor molecules chosen for its photoactivation would have to have absorption maxima below this wavelength, this clearly being impractical. Aromatic azides, however, are capable of photosensitized activation by donors that absorb at longer wavelengths than those absorbed by the azides themselves (14, 15). Photosensitization occurs by various mechanisms (16, 17) in a process that requires the approach of the photosensitizer and acceptor pair to within the collisional range in order for intermolecular exchange of excitation energy to take place (18). The nature of the reactive intermediates generated by photosensitization of aryl azides in different solvents is not completely understood. However, they seem to be reactive in hydrogen-abstraction, dimerization, and insertion reactions (19,20). Since many sensitizers are aromatic apolar molecules, it is likely that they will parallel the distribution of Ina into lipophilic regions of the membrane. This sh...

show abstract

“…Dark reactions are also inferred by comparison of spectrophotometric scans of NAPT solutions done at once and 1 h after photolysis. The reported use of adamantanediazirine (Bayley and Knowles, 1980) to label extrinsic and freely soluble proteins prompted us to investigate carbenes as an alternative photoprobe. The carbene precursor [3H]CDBG was prepared for these studies.…”

Section: Discussionmentioning

confidence: 99%

“…Carbenes generated from hydrophobic diazirines have been used to label the portion of proteins in contact with membrane lipids Bayley and Knowles (1980) have employed a phenyldiazirine derivative of phospholipid to show that at least nine different amino acids of glycophorin were labeled. Diazirines can be activated by longer wavelength light than nitrenes so that damage to structures sensitive to ultraviolet light can be avoided.…”

Section: Introductionmentioning

confidence: 99%

A New Diazirine for Protein Modification by Flash Photolysis: Comparison With an Azide

Johnson

Brown

1986

Photochem & Photobiology

View full text Add to dashboard Cite

Abstract— A new photolabeling agent, N‐[4–(3‐chlorodiazirin‐3‐yl)benzoyl]glycine (CDBG), a carbene generator, was synthesized. The incorporation of its photolytic products into egg white lysozyme was studied using flash photolysis and compared with incorporation of N‐(4‐azido‐2‐nitro‐phenyl)‐2‐amino ethane sulfonate (NAPT) products into lysozyme and bovine pancreatic ribonuclease A. There was considerable additional incorporation of photolysis products into ribonuclease and lysozyme after termination of flash photolysis when NAPT was used but no additional incorporation when CDBG was used. Protein labeled with NAPT retained the label poorly during electrophoresis in sodium dodecyl sulfate. Lysozyme labeled with CDBG lost little label upon electrophoresis. Neither label was well retained during electrophoresis in 8 M urea. Peptic and tryptic peptides from CDBG labeled lysozyme were differentially labeled.

show abstract

Photogenerated reagents for membranes: selective labeling of intrinsic membrane proteins in the human erythrocyte membrane

Cited by 91 publications

References 51 publications

Ebola Virus Inactivation with Preservation of Antigenic and Structural Integrity by a Photoinducible Alkylating Agent

Ebola Virus Inactivation with Preservation of Antigenic and Structural Integrity by a Photoinducible Alkylating Agent

Selective labeling of proteins in biological systems by photosensitization of 5-iodonaphthalene-1-azide.

A New Diazirine for Protein Modification by Flash Photolysis: Comparison With an Azide

Contact Info

Product

Resources

About