Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers

Chu, Shidong; Emwas, Abdul‐Hamid; Lorigan, Gary A.

doi:10.1016/j.jmr.2010.08.012

Cited by 32 publications

(29 citation statements)

References 61 publications

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“…It is interesting that our MD-estimated membrane depths of 5– and 10–Doxyl labels correlate very well with the depths derived from the recent NMR experiments. 13 There is, however, some deviation between the depth of C-14 estimated by MD/X-ray and NMR techniques, Figure 7. We don’t have firm evidence for the origin of this deviation, but it could be related to the variations in lipid compositions used in different sets of experiments (DMPC and PSPC vesicles in the case of X-ray 9,18 and NMR, 13 respectively) and MD simulations (POPC bilayer).…”

Section: Resultsmentioning

confidence: 98%

Molecular Dynamics Simulations of Depth Distribution of Spin-Labeled Phospholipids within Lipid Bilayer

Kyrychenko

Ladokhin

2013

J. Phys. Chem. B

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Spin-labeled lipids are commonly used as fluorescence quenchers in studies of membrane penetration of dye-labeled proteins and peptides using depth-dependent quenching. Accurate calculations of depth of the fluorophore rely on the use of several spin labels placed in the membrane at various positions. The depth of the quenchers (spin probes) has to be determined independently, however; experimental determination of transverse distributions of spin probe depths is difficult. In this article, we use molecular dynamics (MD) simulations to study the membrane behavior and depth distributions of spin–labeled phospholipids in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer. To probe different depths within the bilayer, a series containing five Doxyl-labeled lipids (n-Doxyl PC) has been studied, in which a spin moiety was covalently attached to n–th carbon atoms (where n=5, 7, 10, 12 and 14) of the sn-2 stearoyl chain of the host phospholipid. Our results demonstrate that the chain–attached spin labels are broadly distributed across the model membrane and their environment is characterized by a high degree of mobility and structural heterogeneity. Despite the high thermal disorder, the depth distributions of the Doxyl labels were found to correlate well with their attachment positions, indicating that the distribution of the spin label within the model membrane is dictated by the depth of the n–th lipid carbon atom and not by intrinsic properties of the label. In contrast, a much broader and heterogeneous distribution was observed for a headgroup–attached Tempo spin label of Tempo–PC lipids. MD simulations reveal that, due to the hydrophobic nature, a Tempo moiety favors partitioning from the headgroup region deeper into the membrane. Depending on the concentration of Tempo-PC lipids, the probable depth of the Tempo moiety could span a range from 14.4 Å to 18.2 Å from the membrane center. Comparison of the MD–estimated immersion depths of Tempo and n-Doxyl labels with their suggested experimental depth positions allows us to review critically the possible sources of error in depth-dependent fluorescence quenching studies.

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Section: Resultsmentioning

confidence: 98%

Molecular Dynamics Simulations of Depth Distribution of Spin-Labeled Phospholipids within Lipid Bilayer

Kyrychenko

Ladokhin

2013

J. Phys. Chem. B

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“…We investigated the TOTAPOL and AMUPol distribution in lipid membranes by measuring the lipid 1 H and 13 C spectral intensities at ambient temperature to observe distance-dependent PRE (Buffy relaxation due to the biradical broadens the NMR et al, 2003; Chu et al, 2010). Faster nuclear-spin T 2 signals and reduces peak intensities.…”

Section: Resultsmentioning

confidence: 99%

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

et al. 2016

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Although dynamic nuclear polarization (DNP) has dramatically enhanced solid-state NMR spectral sensitivities of many synthetic materials and some biological macromolecules, recent studies of membrane-protein DNP using exogenously doped paramagnetic radicals as polarizing agents have reported varied and sometimes surprisingly limited enhancement factors. This motivated us to carry out a systematic evaluation of sample preparation protocols for optimizing the sensitivity of DNP NMR spectra of membrane-bound peptides and proteins at cryogenic temperatures of ~110 K. We show that mixing the radical with the membrane by direct titration instead of centrifugation gives a significant boost to DNP enhancement. We quantify the relative sensitivity enhancement between AMUPol and TOTAPOL, two commonly used radicals, and between deuterated and protonated lipid membranes. AMUPol shows ~4 fold higher sensitivity enhancement than TOTAPOL, while deuterated lipid membrane does not give net higher sensitivity for the membrane peptides than protonated membrane. Overall, a ~100 fold enhancement between the microwave-on and microwave-off spectra can be achieved on lipid-rich membranes containing conformationally disordered peptides, and absolute sensitivity gains of 105–160 can be obtained between low-temperature DNP spectra and high-temperature non-DNP spectra. We also measured the paramagnetic relaxation enhancement of lipid signals by TOTAPOL and AMUPol, to determine the depths of these two radicals in the lipid bilayer. Our data indicate a bimodal distribution of both radicals, a surface-bound fraction and a membrane-bound fraction where the nitroxides lie at ~10 Å from the membrane surface. TOTAPOL appears to have a higher membrane-embedded fraction than AMUPol. These results should be useful for membrane-protein solid-state NMR studies under DNP conditions and provide insights into how biradicals interact with phospholipid membranes.

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“…1), allowing examination of a shallower interfacial membrane penetration. This brought demand for a better characterization of the depth of the lipid-attached doxyl and TEMPO groups, which was achieved with various experimental approaches (17-22) and later with MD simulations (23). Because practical applications of DDFQ require relatively high concentration of quenchers, it was important to verify that the presence of multiple bulky quenching groups does not perturb the bilayer.…”

Section: Overview Of Lipid-attached Quenchersmentioning

confidence: 99%

Measuring membrane penetration with depth-dependent fluorescence quenching: Distribution analysis is coming of age

Ladokhin

2014

Biochimica et Biophysica Acta (BBA) - Biomembranes

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Depth-dependent fluorescence quenching by lipid-attached quenchers (e.g., bromine atoms or doxyl groups) is an important tool for determining the penetration of proteins and peptides into lipid bilayers. Extracting quantitative information and accurate calculations of the depth of the fluorophore are complicated by thermal disorder, resulting in broad distributions of the transverse positions of both quenchers and fluorophores. Twenty-one years ago a methodology called Distribution Analysis (DA) was introduced, based on the emerging view of the complexity of the transverse organization of lipid bilayer structure. The method is aimed at extracting quantitative information on membrane penetration, such as position and width of fluorophore's distribution along the depth coordinate and its exposure to the lipid phase. Here we review recent progress in refining the DA method and illustrate its applications to protein-membrane interactions. We demonstrate how basic assumptions of the DA approach can be validated using molecular dynamics simulations and how the precision of depth determination is improved by applying a new protocol based on a combination of steady-state and time-resolved fluorescence quenching. Using the example of the MPER fragment of the membrane-spanning domain of the HIV-1 gp41 fusion protein, we illustrate how DA applications and computer simulations can be used together to reveal the molecular organization of a protein-membrane complex.

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Solid-state NMR paramagnetic relaxation enhancement immersion depth studies in phospholipid bilayers

Cited by 32 publications

References 61 publications

Molecular Dynamics Simulations of Depth Distribution of Spin-Labeled Phospholipids within Lipid Bilayer

Molecular Dynamics Simulations of Depth Distribution of Spin-Labeled Phospholipids within Lipid Bilayer

Efficient DNP NMR of membrane proteins: sample preparation protocols, sensitivity, and radical location

Measuring membrane penetration with depth-dependent fluorescence quenching: Distribution analysis is coming of age

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