Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Chafia Hejase Trad; William James; Anita Bhardwaj; D. Allan Butterfield

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

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Chafia Hejase Trad
, William James
, Anita Bhardwaj
, D. Allan Butterfield

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ³-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

Original language	English
Pages (from-to)	287-299
Number of pages	13
Journal	Journal of Biochemical and Biophysical Methods
Volume	30
Issue number	4
DOIs	https://doi.org/10.1016/0165-022X(95)00016-9
State	Published - Nov 1995

Bibliographical note

Funding Information:
This work was supportedi n part by grantsf rom NSF (AG-10836).

Funding

This work was supportedi n part by grantsf rom NSF (AG-10836).

Funders	Funder number
U.S. Department of Energy Chinese Academy of Sciences Guangzhou Municipal Science and Technology Project Oak Ridge National Laboratory Extreme Science and Engineering Discovery Environment National Science Foundation National Energy Research Scientific Computing Center National Natural Science Foundation of China
National Institute on Aging	P01AG010836
National Institute on Aging

Keywords

EPR
Membrane protein
Methanethiosulfonate
Protein-specific spin labelling

ASJC Scopus subject areas

Biophysics
Biochemistry

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10.1016/0165-022X(95)00016-9

Cite this

@article{d6f64e5317bd4a978985e9ea5039531f,

title = "Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label",

abstract = "Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.",

keywords = "EPR, Membrane protein, Methanethiosulfonate, Protein-specific spin labelling",

author = "Trad, \{Chafia Hejase\} and William James and Anita Bhardwaj and Butterfield, \{D. Allan\}",

note = "Funding Information: This work was supportedi n part by grantsf rom NSF (AG-10836).",

year = "1995",

month = nov,

doi = "10.1016/0165-022X(95)00016-9",

language = "English",

volume = "30",

pages = "287--299",

number = "4",

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TY - JOUR

T1 - Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

AU - Trad, Chafia Hejase

AU - James, William

AU - Bhardwaj, Anita

AU - Butterfield, D. Allan

N1 - Funding Information: This work was supportedi n part by grantsf rom NSF (AG-10836).

PY - 1995/11

Y1 - 1995/11

N2 - Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

AB - Electron paramagnetic resonance was used to characterize the first use of a thiol-specific spin label in membranes. Procedures for use of the spin-label, 1-oxyl-2,2,5,5-tetramethyl-Δ3-pyrroline-3-methyl (methanethiosulfonate MTS) covalently attached to membrane proteins in human erythrocyte membranes are reported. The major findings are: (1) MTS was found to be thiol-specific in membranes as it is for soluble proteins; (2) MTS labels ghost proteins in as few as 30 min at room temperature, providing a distinct advantage when sensitive or fragile membranes are to be used; (3) the distribution of the spin label suggests that the major cytoskeletal protein, spectrin, and the major transmembrane protein (Band 3) incorporate the highest percentage of spin label. This procedure expands the tools with which the researcher can investigate the physical state of membrane proteins and its alteration upon interaction of membrane perturbants or in pathological conditions.

KW - EPR

KW - Membrane protein

KW - Methanethiosulfonate

KW - Protein-specific spin labelling

UR - https://www.scopus.com/pages/publications/0028869250

UR - https://www.scopus.com/pages/publications/0028869250#tab=citedBy

U2 - 10.1016/0165-022X(95)00016-9

DO - 10.1016/0165-022X(95)00016-9

M3 - Article

C2 - 8621887

AN - SCOPUS:0028869250

SN - 0165-022X

VL - 30

SP - 287

EP - 299

JO - Journal of Biochemical and Biophysical Methods

JF - Journal of Biochemical and Biophysical Methods

IS - 4

ER -

Selective labeling of membrane protein sulfhydryl groups with methanethiosulfonate spin label

Abstract

Bibliographical note

Funding

Keywords

ASJC Scopus subject areas

Access to Document

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