Membranous Na,K-ATPase from shark salt gland and from pig kidney was spin-labelled on Class I −SH groups in the presence of glycerol, or on Class II −SH groups in the absence of glycerol after prelabelling Class I groups with N-ethyl maleimide. The Class I-labelled preparations retain full enzymatic activity, whereas the Class II-labelled preparations are at least partially inactivated. The polarity of the environment, and the librational dynamics and conformational exchange, of the spin-labelled groups were studied with pulsed electron paramagnetic resonance (EPR) by using electron spin echo envelope modulation (ESEEM) spectroscopy and spin-echo detected (ED) EPR spectroscopy, respectively. 2H-ESEEM spectra of membranes dispersed in D2O reveal that Class I groups of the shark enzyme are more exposed to water than are those of the kidney enzyme or Class II groups of either species, indicating a 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 characterised by a single phase-memory T2M relaxation time. Conventional EPR lineshapes also demonstrate conformational microheterogeneity at low temperatures: the inhomogeneously broadened lines narrow progressively with increasing temperature reaching a pure Lorentzian lineshape 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-labelled –SH groups: Pulsed EPR of Na,K-ATPase / Guzzi, Rita; Bartucci, R.; Sportelli, L.; Esmann, M.; Marsh, D.. - In: BIOCHEMISTRY. - ISSN 0006-2960. - 48(2009), pp. 8343-8354.
Scheda prodotto non validato
Attenzione! I dati visualizzati non sono stati sottoposti a validazione da parte dell'ateneo
|Titolo:||Conformational heterogeneity and spin-labelled –SH groups: Pulsed EPR of Na,K-ATPase|
|Data di pubblicazione:||2009|
|Citazione:||Conformational heterogeneity and spin-labelled –SH groups: Pulsed EPR of Na,K-ATPase / Guzzi, Rita; Bartucci, R.; Sportelli, L.; Esmann, M.; Marsh, D.. - In: BIOCHEMISTRY. - ISSN 0006-2960. - 48(2009), pp. 8343-8354.|
|Appare nelle tipologie:||1.1 Articolo in rivista|