The role of interfacial lipids in stabilizing membrane protein oligomers _ nature _ nature research

Oligomerization of membrane proteins in response to lipid binding has a critical role in many cell-signalling pathways 1 but is often difficult to define 2 or predict 3. P d database Here we report the development of a mass spectrometry platform to determine simultaneously the presence of interfacial lipids and oligomeric stability and to uncover how lipids act as key regulators of membrane-protein association. Database 101 Evaluation of oligomeric strength for a dataset of 125 α-helical oligomeric membrane proteins reveals an absence of interfacial lipids in the mass spectra of 12 membrane proteins with high oligomeric stability. M power database For the bacterial homologue of the eukaryotic biogenic transporters (LeuT 4, one of the proteins with the lowest oligomeric stability), we found a precise cohort of lipids within the dimer interface.


Data recovery from external hard drive Delipidation, mutation of lipid-binding sites or expression in cardiolipin-deficient Escherichia coli abrogated dimer formation. Database join types Molecular dynamics simulation revealed that cardiolipin acts as a bidentate ligand, bridging across subunits. Section 8 database Subsequently, we show that for the Vibrio splendidus sugar transporter SemiSWEET 5, another protein with low oligomeric stability, cardiolipin shifts the equilibrium from monomer to functional dimer. Icd 9 database We hypothesized that lipids are essential for dimerization of the Na +/H + antiporter NhaA from E. Database xampp coli, which has the lowest oligomeric strength, but not for the substantially more stable homologous Thermus thermophilus protein NapA. Database administrator jobs We found that lipid binding is obligatory for dimerization of NhaA, whereas NapA has adapted to form an interface that is stable without lipids. Data recovery joondalup Overall, by correlating interfacial strength with the presence of interfacial lipids, we provide a rationale for understanding the role of lipids in both transient and stable interactions within a range of α-helical membrane proteins, including G-protein-coupled receptors.

Protein oligomers are represented by circles colour-coded according to the number of salt bridges they form, where maximum number of salt bridge is designated to 100% and 0 is marked as 0% and are grouped by oligomeric state (pentamers+ have an oligomeric state ≥5). Database of genomic variants A random horizontal jitter has been applied to all points to aid visualization. Database viewer NhaA and LeuT (outlined in red) are two of the weakest oligomers, having some of the lowest buried surface areas and forming no salt bridges. H data recovery registration code free download The 12 proteins for which mass spectra have been recorded are outlined in green. Database hardware Illustrated are mass spectra of the trimeric ammonia channel AmtB, tetrameric aquaporin AqpZ and pentameric ion channel ELIC. Database roles A larger buried surface area than LeuT and NhaA but an absence of salt bridges make SemiSWEET a stronger dimer than LeuT and NhaA but weaker than the other 12 oligomeric proteins.

a, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin around LeuT. B tree database management system The surface densities represent the most occupied positions from the simulations of the phosphate (orange), glycerol (red) and alkyl tails (purple) particles of cardiolipin. Database file The proposed binding sites at the interface are the only places where cardiolipin shows considerable population density. Data recovery near me b– d, Comparative particle densities of cardiolipin ( b), phosphatidylglycerol ( c) and phosphoethanolamine ( d) at the LeuT dimeric interface, summed over the simulations show no or minimal densities for phosphatidylglycerol and phosphoethanolamine at the cardiolipin-binding site. Database job description Together, a– d show that the proposed binding sites of cardiolipin at the interface are sites of specific bindings. Data recovery 94fbr e, Dimeric structure of LeuT with modelled APT (aminopentanetetrol, aminophospholipids) classes of lipid present in A. Database foreign key aeolicus 52. Database as a service The lipid was drawn in ChemDraw and subsequently modelled by superimposition onto cardiolipin to give the cardiolipin-bound dimeric structure. Iphone 6 data recovery The favourable van der Waals distances show that it is capable of bridging the dimeric entity through the same sets of residues that were found to be critical towards cardiolipin binding, in an endogenous environment lacking cardiolipins. Database google drive f, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin (phosphate group in orange, glycerol in red and alkyl tails in purple) and POPG (in blue) around NhaA dimer interface. Data recovery geek squad As before, the density of cardiolipin is considerably higher than that of phosphatidylglycerol. Database recovery pending However, unlike LeuT, here the difference between the density of cardiolipin and phosphatidylglycerol is lower, suggesting this site has less exclusivity towards cardiolipin than that in LeuT. Data recovery prices Indeed, mass spectrometry analysis shows a heterogenous distribution of lipids with dimeric NhaA, with mostly cardiolipin but some amount of bound phospholipids.

a, Mass spectra of LeuT, liberated from octylglucoside micelles, (green/grey spheres, most abundant charge state highlighted in pale blue), show that the 7.4-kDa lipid adduct (blue/purple head groups) is retained throughout the trap collision energy range (white, blue arrow) of the mass spectrometer. Database sharding b, Mass spectra of LeuT expressed as a fusion protein with eYFP (LeuT–eYFP yellow circles), liberated from octylglucoside micelles, show that the dimer is similarly associated with a 7.4-kDa adduct.

a, Mass spectrum of LeuT liberated from octylglucoside micelles (green head groups) shows low-abundance delipidated monomers (green spheres, 59.3 kDa) and high-abundance lipid-bound dimers (green/black spheres, 126.0 kDa). Database keys with example b, Mass spectrum of LeuT after incubation with neopentyl glycol (NG, orange head-groups) shows only delipidated monomers. Data recovery xfs c, Mass spectrum of LeuT in octylglucoside (OG), after incubation with neopentyl glycol, shows only delipidated monomers. Database management systems 3rd edition d, Mass spectrum recorded after incubation of delipidated LeuT monomers, in octylglucoside, with E. Database engineer salary coli polar lipids (blue/purple head-groups) shows delipidated monomers and lipid-bound dimers. Jstor database e, Mass spectrum recorded after adding dilysocardiolipin (blue head-groups) to delipidated monomeric LeuT in octylglucoside ( c) shows no dimerization in the presence of this lipid.

Extended Data Figure 3: High-energy MS/MS experiment of the 23+ charge state of dimeric LeuT, with the 7.4-kDa adduct, as a function of collision voltage.

Three satellite peaks represent the lipid-bound states arising through the dissociation of the monomer. E m database The naked monomer is highlighted in blue, while the three satellite peaks are assigned to one phospholipid, one cardiolipin and three phospholipid-bound species (red, green and yellow, respectively). Data recovery richmond va Under higher energy, only the cardiolipin-bound species remains, discounting the mathematical possibility of two phospholipid-bound species. Data recovery software Inset shows the isolated 23+ charge state of the lipid bound dimer. Data recovery advisor Presence of bound cardiolipin at a higher energy, over that of phospholipid, indicates a higher binding energy of cardiolipin over the latter, potentially owing to greater ionic and hydrophobic interactions.

Extended Data Figure 4: Site-directed mutagenesis of selected residues at the LeuT dimer interface, resulting mass spectra and molecular dynamics simulations.

a, Mass spectrum of LeuT F488A/Y489A, liberated from octylglucoside micelles, reveals monomeric LeuT (green spheres). Database host name Inset shows the LeuT dimer interface, with key π-stacking interactions (yellow dotted lines, distances labelled in red) and between aromatic residues (purple). Database performance When residues F488 and Y489 (orange arrows) are mutated to alanine, the π-stacking interactions are abolished and LeuT cannot dimerize. Data recovery broken hard drive b, Molecular dynamics simulations of LeuT in an E. Database xe coli lipid bilayer reveal possible binding sites of interfacial phospholipids and cardiolipin (upper panel, viewed from cytoplasmic side of membrane). Database yml mysql The cardiolipin phosphate groups (orange) interact closely with positively charged residues (K376, H377, R506; blue) at the dimer interface. 5 database is locked Phosphoethanolamine (PE) and phosphatidylglycerol (PG) also bind at the dimer interface. Database fundamentals c, Mass spectrum of LeuT expressed in a cardiolipin-deficient E. Database concepts coli strain (BKT22), liberated from octylglucoside micelles, shows monomeric LeuT, implying that cardiolipin is required for LeuT dimerization. Database icon d, Mass spectrum of LeuT K376A/H377A, liberated from octylglucoside micelles, shows monomeric LeuT.

a, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin around LeuT. Database versioning The surface densities represent the most occupied positions from the simulations of the phosphate (orange), glycerol (red) and alkyl tails (purple) particles of cardiolipin. Database 2013 The proposed binding sites at the interface are the only places where cardiolipin shows considerable population density. Database cursor b– d, Comparative particle densities of cardiolipin ( b), phosphatidylglycerol ( c) and phosphoethanolamine ( d) at the LeuT dimeric interface, summed over the simulations show no or minimal densities for phosphatidylglycerol and phosphoethanolamine at the cardiolipin-binding site. Database list Together, a– d show that the proposed binding sites of cardiolipin at the interface are sites of specific bindings. Database queries must be e, Dimeric structure of LeuT with modelled APT (aminopentanetetrol, aminophospholipids) classes of lipid present in A. Database journal aeolicus 52. Data recovery boston The lipid was drawn in ChemDraw and subsequently modelled by superimposition onto cardiolipin to give the cardiolipin-bound dimeric structure. Database connection The favourable van der Waals distances show that it is capable of bridging the dimeric entity through the same sets of residues that were found to be critical towards cardiolipin binding, in an endogenous environment lacking cardiolipins. S memo data recovery f, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin (phosphate group in orange, glycerol in red and alkyl tails in purple) and POPG (in blue) around NhaA dimer interface. Database structure As before, the density of cardiolipin is considerably higher than that of phosphatidylglycerol. Data recovery iso However, unlike LeuT, here the difference between the density of cardiolipin and phosphatidylglycerol is lower, suggesting this site has less exclusivity towards cardiolipin than that in LeuT. Iphone 6 data recovery software Indeed, mass spectrometry analysis shows a heterogenous distribution of lipids with dimeric NhaA, with mostly cardiolipin but some amount of bound phospholipids.

a, Mass spectrum of unmodified SemiSWEET, liberated from tetraethyleneglycolmonooctyl ether (C 8E 4) micelles, reveals SemiSWEET monomers and dimers (black spheres). Cpu z database b, Mass spectrum of deca-His tagged SemiSWEET, liberated from C 8E 4 micelles, reveals SemiSWEET monomers and dimers (green spheres). Data recovery kickass c, High energy MS/MS of unmodified SemiSWEET, liberated from dodecylmaltoside (DDM) micelles, allows isolation of the 6+ charge state (black spheres) of the SemiSWEET monomer (black spheres) bound to endogenous lipids. A database can best be described as Fragmentation of the lipid-bound species leads to loss of either cardiolipin (1,470 ± 26 Da, purple head-groups), one or two neutral phospholipids (each 756 ± 22 Da, blue head-groups), or a positively charged phospholipid. Os x database Trap collision voltages are shown in white inside the blue arrow. Database field d, Mass spectrum of deca-His SemiSWEET, liberated from C 8E 4 micelles and incubated with phosphatidylglycerol (blue head-groups). Data recovery diy phosphatidylglycerol binds to both monomers and dimers (dotted boxes highlight lipid-bound peaks) without substantial preference. Database transaction e, Mass spectrum recorded after incubation in solution of an equimolar ratio of deca-His tagged and untagged SemiSWEET (green and black spheres, respectively), liberated from tetraethyleneglycolmonooctyl ether (C 8E 4) micelles. Data recovery mac hard drive Plot of the percentage abundance of hetero- and homodimers over time (inset), SemiSWEET heterodimers (red trace, peaks highlighted red in mass spectrum) and homodimers (black trace), revealing the solution-phase monomer–dimer equilibrium (PDB accession number: 4QND).

a, Mass spectrum of NhaA, liberated from C 8E 4 micelles, reveals NhaA monomers (green spheres) bound to cardiolipin (purple head-groups) and an ensemble of NhaA dimer species in different lipidation states (highlighted in green). H2 database tutorial b, MS/MS of the 15+ charge state (green) of the NhaA dimer (green/black spheres) bound to two cardiolipin molecules liberated from C 8E 4 micelles. Database interview questions Increasing collision voltage applied to the 2× cardiolipin-bound species leads either to loss of 1 cardiolipin to form NhaA dimers bound to 1 cardiolipin (40 V) or to loss of 2 cardiolipin molecules to form delipidated NhaA dimers, with concomitant generation of NhaA monomers (70 V) and further dissociation of NhaA dimers into monomers (120 V). R studio data recovery free download Trap collision voltages are depicted in white, inside the blue arrow.

a, The basic residues of LeuT that are involved in lipid binding (red box) are conserved across the BATs. Data recovery bad hard drive b, Two views of the superimposed structures of LeuT (PDB accession number: 2A65, black) and SERT (PDB accession number: 5I6Z, light blue) show the differences in the dimer interface. Database field definition Dimer interface helices are highlighted with arrows and coloured (LeuT, green; SERT, red); basic residues responsible for lipid binding in LeuT are shown in yellow mesh. Data recovery windows 7 One of the interface helices in SERT swings away from the interface, negating the possibility of lipid-induced oligomerization, analogous to that proposed for LeuT.

a, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin around LeuT. Nexus 4 data recovery The surface densities represent the most occupied positions from the simulations of the phosphate (orange), glycerol (red) and alkyl tails (purple) particles of cardiolipin. Database version 706 The proposed binding sites at the interface are the only places where cardiolipin shows considerable population density. Cindia data recovery b– d, Comparative particle densities of cardiolipin ( b), phosphatidylglycerol ( c) and phosphoethanolamine ( d) at the LeuT dimeric interface, summed over the simulations show no or minimal densities for phosphatidylglycerol and phosphoethanolamine at the cardiolipin-binding site. Database tutorial Together, a– d show that the proposed binding sites of cardiolipin at the interface are sites of specific bindings. R database packages e, Dimeric structure of LeuT with modelled APT (aminopentanetetrol, aminophospholipids) classes of lipid present in A. Database disk image is malformed aeolicus 52. Windows 8 data recovery software The lipid was drawn in ChemDraw and subsequently modelled by superimposition onto cardiolipin to give the cardiolipin-bound dimeric structure. Database naming standards The favourable van der Waals distances show that it is capable of bridging the dimeric entity through the same sets of residues that were found to be critical towards cardiolipin binding, in an endogenous environment lacking cardiolipins. Data recovery training online f, Particle densities from five repeats of 1-μs CGMD simulations for cardiolipin (phosphate group in orange, glycerol in red and alkyl tails in purple) and POPG (in blue) around NhaA dimer interface. Database query As before, the density of cardiolipin is considerably higher than that of phosphatidylglycerol. Database isolation levels However, unlike LeuT, here the difference between the density of cardiolipin and phosphatidylglycerol is lower, suggesting this site has less exclusivity towards cardiolipin than that in LeuT. Database version control Indeed, mass spectrometry analysis shows a heterogenous distribution of lipids with dimeric NhaA, with mostly cardiolipin but some amount of bound phospholipids.

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