Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated, This provides all excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) call provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. (2)H-ESEEM spectra of membranes dispersed in D(2)O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class 11 groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures ( < 150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T(2M) relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching all almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.

Conformational Heterogeneity and Spin-Labeled -SH Groups: Pulsed EPR Na,K-ATPase / Guzzi, R; Bartucci, R; Sportelli, Luigi; Esmann, M; Marsh, D.. - In: BIOCHEMISTRY. - ISSN 0006-2960. - 48(2009), pp. 8343-8354.

Conformational Heterogeneity and Spin-Labeled -SH Groups: Pulsed EPR Na,K-ATPase

GUZZI R;BARTUCCI R;SPORTELLI, Luigi;
2009

Abstract

Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labeled on class I -SH groups in the presence of glycerol, or on class II -SH groups in the absence of glycerol. The class I-labeled preparations retain full enzymatic activity, whereas the class II-labeled preparations are at least partially inactivated, This provides all excellent testbed on which to demonstrate how advanced electron paramagnetic resonance (EPR) call provide novel information on specific residues in unique environments in a complex, membrane-bound transport system. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labeled groups were studied with pulsed EPR by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. (2)H-ESEEM spectra of membranes dispersed in D(2)O reveal that class I groups of the shark enzyme are more exposed to water than are those of the pig enzyme or class 11 groups of either species, consistent with the more superficial membrane location in the former case. Spin-echo decay curves indicate conformational heterogeneity at low temperatures ( < 150 K), but a more homogeneous conformational state at higher temperatures that is characterized by a single phase-memory T(2M) relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching all almost pure Lorentzian line shape at temperatures of ca. 220 K and above. The inhomogeneous broadening at low temperature is well described by a Gaussian distribution of Lorentzian lines. ED spectra as a function of echo-delay time demonstrate the onset of rapid librational motions of appreciable amplitude, and slower conformational exchange, at temperatures above 220 K. These motions could drive transitions between the different conformational substates, which are frozen in at lower temperatures but contribute to the pathways between the principal enzymatic intermediates at higher temperatures.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/20.500.11770/153179
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