Measuring 13C–2D Dipolar Couplings with a Universal REDOR Dephasing Curve

Gullion, Terry

doi:10.1006/jmre.2000.2164

Cited by 29 publications

(27 citation statements)

References 19 publications

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“…Whereas in this and in our previous studies 9 -11 we used the technique of lineshape analysis in order to determine these couplings, in view of the simplicity of our 15 N spectra, these couplings can no longer be easily obtained in this way in more complex systems. Therefore, Sack et al 20 and Gullion 21 proposed REDOR-type techniques such as CPL-REDOR in order to obtain these couplings under MAS conditions, and Schmidt-Rohr and co-workers the RIDER technique.…”

Section: Discussionmentioning

confidence: 98%

1H/15N NMR chemical shielding, dipolar15N,2H coupling and hydrogen bond geometry correlations in a novel series of hydrogen-bonded acid-base complexes of collidine with carboxylic acids

et al. 2001

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Section: Discussionmentioning

confidence: 98%

1H/15N NMR chemical shielding, dipolar15N,2H coupling and hydrogen bond geometry correlations in a novel series of hydrogen-bonded acid-base complexes of collidine with carboxylic acids

et al. 2001

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“…The H/D exchange procedure exploits the modified REDOR experiment described by Gullion (40), which reintroduces the dipolar interaction between a spin half-nucleus such as 13 C and a spin 1 nucleus such as 2 H. The recoupled dipolar interaction is observed as a reduction of the peak intensity, S, from the observed nucleus relative to the peak intensity, S 0 , measured in a control experiment. The drop in the peak intensity, which can be normalized as S/S 0 , is related to the through-space internuclear distance r IS between the two nuclei and to the experimental echo period t e during which dipolar recoupling is allowed to occur.…”

Section: Effect Of Ca 2ϩ -Atpase On Structure Of Phospholambanmentioning

confidence: 99%

“…The membranes were suspended in solution in 90% D 2 O, incubated at 4°C for 15 min, centrifuged to a pellet, flash-frozen, and maintained at Ϫ40°C during 13 C, 2 H-REDOR NMR analysis to prevent further H/D exchange. The echo time, t e , was set to 3.5 ms, which is predicted to produce the maximum possible dephasing for an internuclear distance of 2.0 Å (40 The results of the H/D exchange experiment are shown in Fig. 3, B-D.…”

Section: Effect Of Ca 2ϩ -Atpase On Structure Of Phospholambanmentioning

confidence: 99%

Solid-state NMR Reveals Structural Changes in Phospholamban Accompanying the Functional Regulation of Ca2+-ATPase

Hughes

Middleton

2003

Journal of Biological Chemistry

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Calcium transport across the sarcoplasmic reticulum of cardiac myocytes is regulated by a reversible inhibitory interaction between the Ca 2؉ -ATPase and the small transmembrane protein phospholamban (PLB). A nullcysteine analogue of PLB, containing isotope labels in the transmembrane domain or cytoplasmic domain, was reconstituted into membranes in the absence and presence of the SERCA1 isoform of Ca 2؉ -ATPase for structural investigation by cross-polarization magic-angle spinning (CP-MAS) NMR. PLB lowered the maximal hydrolytic activity of SERCA1 and its affinity for calcium in membrane preparations suitable for structural analysis by NMR. switches from an ␣-helix in pure lipid membranes to a more extended structure in the presence of SERCA1, which may reflect local structural distortions which change the orientations of the transmembrane and cytoplasmic domains. These results suggest that Ca 2؉ -ATPase has a long-range effect on the structure of PLB around residue 25, which promotes the functional association of the two proteins. Phospholamban (PLB)1 is a 52-amino acid membrane-spanning protein, which is expressed predominantly in the sarcoplasmic reticulum of cardiac myocytes (1). The primary physiological function of PLB is to regulate the active transport of calcium ions into the sarcoplasmic reticulum lumen via an inhibitory association with SERCA2a, the cardiac isoform of Ca 2ϩ -ATPase (2, 3). PLB is believed to bind to the calcium-free conformation of SERCA2a (4), and exerts its inhibitory action by reducing the apparent calcium affinity of the enzyme (5). In response to ␤-adrenergic stimulation, PLB is phosphorylated at Ser 16 and Thr 17 by cAMP-dependent kinase and calcium/calmodulin-dependent kinase, respectively. PLB phosphorylation relieves SERCA2a inhibition, which increases the rate of calcium uptake into the sarcoplasmic reticulum and results in the accelerated relaxation of cardiac muscle (2). Mechanistic failures in the reversibility of SERCA2a inhibition, or overexpression of PLB relative to SERCA2a, are linked to the disruption of calcium homeostasis in cardiac cells and may contribute to cardiovascular disorders such as congestive heart failure (6).The exact nature of the interaction between PLB and SERCA enzymes is a subject of debate. PLB readily self-associates to form a homopentamer within the lipid bilayer (7,8), with a dynamic equilibrium believed to exist between the oligomeric and monomeric state of the protein (9). Most evidence suggests that it is the monomeric form of PLB that binds to and inhibits SERCA, with the pentamer acting as a reservoir from which monomers dissociate (5, 8, 10 -12). In reconstitution studies, the molar stoichiometry of PLB to SERCA required for maximal regulation of calcium transport is between 3:1 and 15:1 (12, 13), whereas a fluorescence energy transfer study has suggested that two PLB monomers interact with two Ca 2ϩ ATPase molecules to form a heterodimer (14). In the latter study, it was found that both PLB molecules must be phosphorylated in order t...

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“…Good progress has been made for the case of 13 Cobserve REDOR experiments involving 2 D (24-29). The use of phase-modulated 2 D pulses has provided a means to extract 13 C-2 D dipolar couplings (26,27), and a more recent report shows that the 13 C-2 D REDOR experiment can be described by a universal dipolar dephasing curve when a 2 D composite pulse is used (29).…”

Section: Introductionmentioning

confidence: 99%