Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

Bartucci, Rosa; Guzzi, Rita; Esmann, Mikael; Marsh, Derek

doi:10.1016/j.bpj.2014.07.057

Cited by 11 publications

(11 citation statements)

References 43 publications

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“…Interestingly, there is precedent for protein–lipid interfaces promoting water access to a higher degree than bulk lipid phases. 35 Alternatively, increases in local charge can accelerate H/DX in proteins. 36 Regardless of the source of the differences, the H/DX MS data clearly confirm the role of the A-helix, β 1,2, and nearby F′/G′-regions as the strongest membrane anchors in the nanodisc platform.…”

Section: Discussionmentioning

confidence: 99%

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

et al. 2016

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Membrane-bound cytochrome P4503A4 (CYP3A4) is the major source of enzymatic drug metabolism. Although several structural models of CYP3A4 in various ligand complexes are available, none includes a lipid bilayer. Details of the effects of the membrane on protein dynamics and solvation, and access channels for ligands, remain uncertain. H/D exchange mass spectrometry (H/DXMS) with ligand free CYP3A4 containing a deletion of residues 3–12, compared to that of the full length wild type, in lipid nanodiscs afforded 91% sequence coverage. Deuterium exchange was fast in the F- and G-helices, HI loop, and C-terminal loop. In contrast, there is very low exchange in the F′-and G′-helices. The results are consistent with the overall membrane orientation of CYP3A4 suggested by published MD simulations and spectroscopic results, and the solvent accessibility of the F/G loop suggests that it is not deeply membrane-embedded. Addition of ketoconazole results in only modest, but global, changes in solvent accessibility. Interestingly, with ketoconazole bound some peptides become less solvent accessible or dynamic, including the F- and G-helices, but several peptides demonstrate modestly increased accessibility. Differential scanning calorimetry (DSC) of CYP3A4-nanodiscs suggests membrane-induced stabilization compared to that of aggregated CYP3A4 in buffer, and this stabilization is enhanced upon addition of the ligand ketoconazole. This ligand-induced stabilization is accompanied by a very large increase in ΔH for CYP3A4 denaturation in nanodiscs, possibly due to increased CYP3A4–membrane interactions. Together, the results suggest a distinct orientation of CYP3A4 on the lipid membrane, and they highlight likely solvent access channels, which are consistent with several MD simulations.

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Section: Discussionmentioning

confidence: 99%

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

et al. 2016

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“…Since the ordering effect of the lipid head group has been attributed to bilayer dehydration 53 , we used ESEEM spectroscopy to find out whether the peptide-induced ordering of anionic vesicles is possibly related to membrane dehydration, as suggested as a general fusion mechanism of other class I fusion peptides 46,47 . ESEEM has been successfully applied to examine the penetration depth profile of deuterium-substituted molecules, such as water, glycerol, and sugar inside lipid bilayers [54][55][56][57] as well as to investigate the water permeation at protein/peptide-lipid interface of membrane-interacting peptides and membrane proteins [58][59][60] . Here we used a D 2 O-containing buffer to probe the deuterium environment surrounding the spin labels DOPTC, 5-PCSL, and 16-PCSL ( Supplementary Fig.…”

Section: Dehydration Of Popc/popg Membranesmentioning

confidence: 99%

SARS-CoV fusion peptides induce membrane surface ordering and curvature

Basso

Vicente

Crusca

et al. 2016

Sci Rep

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Viral membrane fusion is an orchestrated process triggered by membrane-anchored viral fusion glycoproteins. The S2 subunit of the spike glycoprotein from severe acute respiratory syndrome (SARS) coronavirus (CoV) contains internal domains called fusion peptides (FP) that play essential roles in virus entry. Although membrane fusion has been broadly studied, there are still major gaps in the molecular details of lipid rearrangements in the bilayer during fusion peptide-membrane interactions. Here we employed differential scanning calorimetry (DSC) and electron spin resonance (ESR) to gather information on the membrane fusion mechanism promoted by two putative SARS FPs. DSC data showed the peptides strongly perturb the structural integrity of anionic vesicles and support the hypothesis that the peptides generate opposing curvature stresses on phosphatidylethanolamine membranes. ESR showed that both FPs increase lipid packing and head group ordering as well as reduce the intramembrane water content for anionic membranes. Therefore, bending moment in the bilayer could be generated, promoting negative curvature. The significance of the ordering effect, membrane dehydration, changes in the curvature properties and the possible role of negatively charged phospholipids in helping to overcome the high kinetic barrier involved in the different stages of the SARS-CoV-mediated membrane fusion are discussed.

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“…SDSL and ESEEM spectroscopy have been used to study the supermolecular structure of biological systems, the penetration depth of water into the membrane and in KcsA K + channels, localization of proteins or lipids in membranes, and protein folding (Carmieli et al, 2006; Cieslak et al, 2010) (Bartucci, Guzzi, Esmann, & Marsh, 2014; Dzuba & Raap, 2013; Matalon, Faingold, Eisenstein, Shai, & Goldfarb, 2013). …”

Section: Introductionmentioning

confidence: 99%

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

Liu

Mayo

Sahu

et al. 2015

Methods in Enzymology

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Revealing detailed structural and dynamic information of membrane embedded or associated proteins is challenging due to their hydrophobic nature which makes NMR and X-ray crystallographic studies challenging or impossible. Electron paramagnetic resonance (EPR) has emerged as a powerful technique to provide essential structural and dynamic information for membrane proteins with no size limitations in membrane systems which mimic their natural lipid bilayer environment. Therefore, tremendous efforts have been devoted toward the development and application of EPR spectroscopic techniques to study the structure of biological systems such as membrane proteins and peptides. This chapter introduces a novel approach established and developed in the Lorigan lab to investigate membrane protein and peptide local secondary structures utilizing the pulsed EPR technique electron spin echo envelope modulation (ESEEM) spectroscopy. Detailed sample preparation strategies in model membrane protein systems and the experimental setup are described. Also, the ability of this approach to identify local secondary structure of membrane proteins and peptides with unprecedented efficiency is demonstrated in model systems. Finally, applications and further developments of this ESEEM approach for probing larger size membrane proteins produced by over-expression systems are discussed.

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Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

Cited by 11 publications

References 43 publications

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

Membrane Interactions, Ligand-Dependent Dynamics, and Stability of Cytochrome P4503A4 in Lipid Nanodiscs

SARS-CoV fusion peptides induce membrane surface ordering and curvature

Determining the Secondary Structure of Membrane Proteins and Peptides Via Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy

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