Saturation transfer electron spin resonance of Ca2+-ATPase covalently spin-labeled with β-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels

L. I. Horvath, L. Dux, H. O. Hankovszky, K. Hideg, D. Marsh

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Abstract

The Ca2+-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide β-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the α-carbon, to enhance further the electrophilic character of the β-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2+-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the line-widths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer line-height ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the line-height ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the line-height ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2+-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.

Original languageEnglish
Pages (from-to)231-241
Number of pages11
JournalBiophysical Journal
Volume58
Issue number1
Publication statusPublished - 1990

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Calcium-Transporting ATPases
Electron Spin Resonance Spectroscopy
Ketones
Sarcoplasmic Reticulum
Carbon
Spin Labels
Proteins
Ethylmaleimide
Membranes
Hydrodynamics
nitroxide maleimide
Electrons
maleimide

ASJC Scopus subject areas

  • Biophysics

Cite this

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title = "Saturation transfer electron spin resonance of Ca2+-ATPase covalently spin-labeled with β-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels",
abstract = "The Ca2+-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide β-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the α-carbon, to enhance further the electrophilic character of the β-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2+-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the line-widths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer line-height ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the line-height ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the line-height ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2+-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.",
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T1 - Saturation transfer electron spin resonance of Ca2+-ATPase covalently spin-labeled with β-substituted vinyl ketone- and maleimide-nitroxide derivatives. Effects of segmental motion and labeling levels

AU - Horvath, L. I.

AU - Dux, L.

AU - Hankovszky, H. O.

AU - Hideg, K.

AU - Marsh, D.

PY - 1990

Y1 - 1990

N2 - The Ca2+-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide β-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the α-carbon, to enhance further the electrophilic character of the β-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2+-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the line-widths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer line-height ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the line-height ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the line-height ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2+-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.

AB - The Ca2+-ATPase in native sarcoplasmic reticulum membranes was selectively spin-labeled for saturation transfer electron spin resonance (ESR) studies by prelabeling with N-ethylmaleimide and by using low label/protein ratios. Results with the nitroxide derivative of the standard sulphydryl-modifying reagent, maleimide, were compared with a series of six novel nitroxide β-substituted vinyl aryl ketone derivatives which differed (with two exceptions) in the substituent at the ketone position. The two exceptions had a different electron withdrawing group at the α-carbon, to enhance further the electrophilic character of the β-carbon. Although differing in their reactivity, all the conjugated unsaturated ketone nitroxide derivatives displayed saturation transfer ESR spectra indicative of much slower motion than did the maleimide derivative. The saturation transfer ESR spectra of maleimide-labeled Ca2+-ATPase therefore most likely contain substantial contributions from segmental motion of the labeled group. The effects of the level of spin labeling were also investigated. With increasing degree of spin label incorporation, the line-widths of the conventional ESR spectrum progressively increased and the intensity of the saturation transfer spectrum dropped dramatically, as a result of increasing spin-spin interactions. The hyperfine splittings of the conventional spectrum and the outer line-height ratios of the saturation transfer spectrum remained relatively unchanged. Extrapolation back to zero labeling level yielded comparable values for the effective rotational correlation times deduced from the saturation transfer spectrum intensities and from the line-height ratios, for the vinyl ketone label. For the maleimide label the extrapolated values from the integral are significantly lower than those from the line-height ratios, probably because of the segmental motion. Comparison is made of the effective rotational correlation time for the vinyl ketone label with the predictions of hydrodynamic models for the protein diffusion, in a discussion of the aggregation state of the Ca2+-ATPase in the native sarcoplasmic reticulum membrane. The implications for the study of protein rotational diffusion and segmental motion, and of the proximity relationships between labeled groups, using saturation transfer ESR spectroscopy are discussed.

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