Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Esmann, Mikael; Marsh, Derek

doi:10.1073/pnas.89.16.7606

Cited by 8 publications

(5 citation statements)

References 20 publications

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“…Both the conventional and saturationtransfer ESR spectra of the squid system indicate that there is no rapid segmental motion of the spin-label, in agreement with this expectation. At the pH used for labeling, it is likely that only cysteine residues on the protein are labeled, in common with studies on other proteins using this label (Esmann et al, 1989(Esmann et al, , 1990Horvdth et al, 1990;Esmann & Marsh, 1992). Comparison of the ESR spectra from the photoreceptor membrane with those from purified rhodopsin reconstituted in phosphatidylcholine confirms, as expected, that only the protein is labeled.…”

Section: Methodssupporting

confidence: 67%

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes

Ryba¹,

Hoon²,

Findlay³

et al. 1993

Biochemistry

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Treatment of outer segment membranes from Loligo forbesi with endoprotease-V8 from Staphylococcus aureus results in cleavage of the C-terminal extension of the squid rhodopsin, with accompanying reduction of the apparent molecular weight from 47,000 to 36,000 on sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Negative-stain electron microscopy of the intact membranes shows that small clusters of the rhodopsin C-termini form structures extending from the membrane surface and that these are absent after protease treatment. Fourier transform infrared spectra of the amide I band of the protein indicate that removal of the C-terminal extension increases the relative alpha-helical content of squid rhodopsin to a level comparable to that for bovine rhodopsin in disk membranes, and to an extent which suggests that the alpha-helical structure lies mainly in the M(r) 36,000 (transmembrane) section of the protein. Saturation-transfer electron spin resonance (ESR) spectroscopy of the spin-labeled protein reveals that the rotational diffusion of squid rhodopsin in outer segment membranes that have been extensively washed with urea to remove peripheral proteins is much slower than that of bovine rhodopsin in rod outer segment disk membranes. This reduction in rotational mobility is also found with purified squid rhodopsin reconstituted in egg phosphatidylcholine and in urea-washed outer segment membranes which have been treated with endoprotease-V8 to remove the C-terminal extension of squid rhodopsin. In the latter case, the saturation-transfer ESR spectra are virtually identical to those of the non-proteolyzed membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodssupporting

confidence: 67%

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes

Ryba¹,

Hoon²,

Findlay³

et al. 1993

Biochemistry

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“…Techniques with sufficiently rapid temporal resolution are electron spin resonance and NMR, which have resolutions of 10-8 and 10's, respectively (Gennis, 1989). Esmann and Marsh (1992) measured Ds of Na+-K+-ATPase in a lipid dispersion at one concentration, which is not enough for a complete test of the theory.…”

Section: Discussionmentioning

confidence: 99%

“…The short time scale for Ds, t << 10-' s, precludes its measurement by FPR or PER. Techniques fast enough to measure Ds exist (Konig et al, 1992;Esmann and Marsh, 1992), and Esmann and Marsh have measured the short-time diffusivity of the Na+,K+-ATPase in a protein-lipid dispersion (Esmann and Marsh, 1992). To our knowledge, the short time diffusivity of a protein has not been measured in a bilayer phospholipid membrane.…”

Section: Short-time Tracer Diffusionmentioning

confidence: 99%

Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes

Bussell

Koch

Hammer

1995

Biophysical Journal

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Tracer diffusion coefficients of integral membrane proteins (IMPs) in intact plasma membranes are often much lower than those found in blebbed, organelle, and reconstituted membranes. We calculate the contribution of hydrodynamic interactions to the tracer, gradient, and rotational diffusion of IMPs in plasma membranes. Because of the presence of immobile IMPs, Brinkman's equation governs the hydrodynamics in plasma membranes. Solutions of Brinkman's equation enable the calculation of short-time diffusion coefficients of IMPs. There is a large reduction in particle mobilities when a fraction of them is immobile, and as the fraction increases, the mobilities of the mobile particles continue to decrease. Combination of the hydrodynamic mobilities with Monte Carlo simulation results, which incorporate excluded area effects, enable the calculation of long-time diffusion coefficients. We use our calculations to analyze results for tracer diffusivities in several different systems. In erythrocytes, we find that the hydrodynamic theory, when combined with excluded area effects, closes the gap between existing theory and experiment for the mobility of band 3, with the remaining discrepancy likely due to direct obstruction of band 3 lateral mobility by the spectrin network. In lymphocytes, the combined hydrodynamic-excluded area theory provides a plausible explanation for the reduced mobility of sIg molecules induced by binding concanavalin A-coated platelets. However, the theory does not explain all reported cases of "anchorage modulation" in all cell types in which receptor mobilities are reduced after binding by concanavalin A-coated platelets. The hydrodynamic theory provides an explanation of why protein lateral mobilities are restricted in plasma membranes and why, in many systems, deletion of the cytoplasmic tail of a receptor has little effect on diffusion rates. However, much more data are needed to test the theory definitively. We also predict that gradient and tracer diffusivities are the same to leading order. Finally, we have calculated rotational diffusion coefficients in plasma membranes. They decrease less rapidly than translational diffusion coefficients with increasing protein immobilization, and the results agree qualitatively with the limited experimental data available.

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“…2 , lower panel , and 3 ). Rapid diffusion within the lipid membrane plane ( 32 , 33 ) thus provides efficient two-dimensional channeling of inhibitor to the Na,K-ATPase before binding. Spectra of spin-labeled bufalins B1 , B4 , B5 , B6 , and B7 and cinobufagins C4 and C8 reveal a motionally restricted component of inhibitors localized at the lipid-protein interface.…”

Section: Discussionmentioning

confidence: 99%

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

Aloi

Guo

Guzzi

et al. 2021

Biophysical Journal

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Spin labels based on cinobufagin, a specific inhibitor of the Na,K-ATPase, have proved valuable tools to characterize the binding site of cardiotonic steroids (CTSs), which also constitutes the extracellular cation pathway. Because existing literature suggests variations in the physiological responses caused by binding of different CTSs, we extended the original set of spin-labeled inhibitors to the more potent bufalin derivatives. Positioning of the spin labels within the Na,K-ATPase site was defined and visualized by molecular docking. Although the original cinobufagin labels exhibited lower affinity, continuouswave electron paramagnetic resonance spectra of spin-labeled bufalins and cinobufagins revealed a high degree of pairwise similarity, implying that these two types of CTS bind in the same way. Further analysis of the spectral lineshapes of bound spin labels was performed with emphasis on their structure (PROXYL vs. TEMPO), as well as length and rigidity of the linkers. For comparable structures, the dynamic flexibility increased in parallel with linker length, with the longest linker placing the spin label at the entrance to the binding site. Temperature-related changes in spectral lineshapes indicate that six-membered nitroxide rings undergo boat-chair transitions, showing that the binding-site cross section can accommodate the accompanying changes in methyl-group orientation. D 2 O-electron spin echo envelope modulation in pulse-electron paramagnetic resonance measurements revealed high water accessibilities and similar polarity profiles for all bound spin labels, implying that the vestibule leading to steroid-binding site and cation-binding sites is relatively wide and water-filled.

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Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Cited by 8 publications

References 20 publications

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes

Rhodopsin mobility, structure, and lipid-protein interaction in squid photoreceptor membranes

Effect of hydrodynamic interactions on the diffusion of integral membrane proteins: diffusion in plasma membranes

Geometry and water accessibility of the inhibitor binding site of Na+-pump: Pulse- and CW-EPR study

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