Investigating Structural Changes in the Lipid Bilayer upon Insertion of the Transmembrane Domain of the Membrane-Bound Protein Phospholamban Utilizing 31P and 2H Solid-State NMR Spectroscopy

Dave, Paresh C.; Tiburu, Elvis K.; Damodaran, Krishnan; Lorigan, Gary A.

doi:10.1016/s0006-3495(04)74224-1

Cited by 49 publications

(57 citation statements)

References 70 publications

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“…In particular, membrane association has been detected by using 2 H and 31 P solid-state NMR with cytoplasmic domain peptides and full-length versions of wt-PLN by the Middleton (19,20) and Lorigan (21,22) groups. The interactions with the membranes or membrane-mimicking systems induce a stable ␣-helical conformation.…”

Section: Discussionmentioning

confidence: 99%

“…This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues [17][18][19][20][21], and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3).…”

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confidence: 99%

“…Recent spectroscopic data from the Middleton (19,20) and Lorigan (21,22) groups suggest that the membrane association of the cytoplasmic domain of PLN also is present in pentameric wt-PLN. These data are in disaccord with the continuous helix conformation, the extended helix/sheet model, and the bellflower structure.…”

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confidence: 99%

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Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

100

126

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Phospholamban (PLN) regulates calcium translocation within cardiac myocytes by shifting sarco(endo)plasmic reticulum Ca 2؉ -ATPase (SERCA) affinity for calcium. Although the monomeric form of PLN (6 kDa) is the principal inhibitory species, recent evidence suggests that the PLN pentamer (30 kDa) also is able to bind SERCA. To date, several membrane architectures of the pentamer have been proposed, with different topological orientations for the cytoplasmic domain: (i) extended from the bilayer normal by 50 -60°; (ii) continuous ␣-helix tilted 28°relative to the bilayer normal; (iii) pinwheel geometry, with the cytoplasmic helix perpendicular to the bilayer normal and in contact with the surface of the bilayer; and (iv) bellflower structure, in which the cytoplasmic domain helix makes Ϸ20°angle with respect to the membrane bilayer normal. Using a variety of cell membrane mimicking systems (i.e., lipid vesicles, oriented lipid bilayers, and detergent micelles) and a combination of multidimensional solution/solidstate NMR and EPR spectroscopies, we tested the different structural models. We conclude that the pinwheel topology is the predominant conformation of pentameric PLN, with the cytoplasmic domain interacting with the membrane surface. We propose that the interaction with the bilayer precedes SERCA binding and may mediate the interactions with other proteins such as protein kinase A and protein phosphatase 1.Ca 2ϩ -ATPase ͉ EPR ͉ membrane protein ͉ solid-state NMR ͉ protein dynamics C alcium translocation into the sarcoplasmic reticulum of cardiac myocytes is controlled by the sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA). Phospholamban (PLN) regulates the activity of SERCA by shifting the apparent calcium affinity for the enzyme. This activity is relieved by phosphorylation of PLN at Ser-16 and/or Thr-17 and high calcium concentration within the cytosol. Wild-type PLN (wt-PLN) forms stable homopentamers in lipid bilayers and in detergent micelles, where each monomer is composed of a helical cytoplasmic domain (residues 1-16), a semiflexible loop (residues 17-21), and a helical transmembrane domain (residues 22-52) (1, 2). Mutagenesis, molecular biology, and in vivo studies revealed that the PLN pentamer depolymerizes into active monomers that bind and inhibit SERCA (3). Similar conclusions were reached by in vitro fluorescence studies (4). Recently, Young and coworkers (5) have reported a cocrystal formed by SERCA and PLN pentamer, suggesting that the pentameric species also is able to bind SERCA. Furthermore, Jones and coworkers (6) hypothesized that the PLN pentamer may act as a chloride ion channel, which is supported by the bellflower structure recently determined by Oxenoid and Chou (2).There are four principal proposed structural models of pentameric wt-PLN, which differ primarily in the topology of the more dynamic cytoplasmic domain. In each of these models (shown in Fig. 1), residues 32-52 are in a coiled helix approximately parallel to the bilayer normal. The first model (extended helix/sheet ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Spectroscopic validation of the pentameric structure of phospholamban

Traaseth¹,

Verardi

Torgersen

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

100

126

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“…42 The order parameters were a little smaller than those for the pure POPC_d 31 / cholesterol bilayers, which means that the insertion of the peptides into the bilayers enables the acyl chains to move more freely than in the POPC_d 31 bilayer/cholesterol bilayers.…”

Section: Resultsmentioning

confidence: 93%

Effect of Cholesterol on the Phase Change of Lipid Membranes by Antimicrobial Peptides

Choi¹,

Kim²

2014

Bulletin of the Korean Chemical Society

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Membrane disruption by an antimicrobial peptide (AMP) was investigated by measuring the 2 H solid-state nuclear magnetic resonance spectra of 1-palmitoyl-d 31 -2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC_d 31 ) in mixtures of POPC_d 31 /cholesterol and either magainin 2 or aurein 3.3 deposited on thin cover-glass plates. The line shapes of the experimental 2 H solid-state nuclear magnetic resonance (SSNMR) spectra were best simulated by assuming the coexistence of a mosaic spread of bilayers containing pore structures and a fasttumbling isotropic phase or a hexagonal phase. Within a few days of incubation in a hydration chamber, an isotropic phase and a pore structure were induced by magainin 2, while in case of aurein 3.3 only an isotopic phase was induced in the presence of a bilayer phase. After an incubation period of over 100 days, alignment of the bilayers increased and the amount of the pore structure decreased in case of magainin 2. In contrast with magainin 2, aurein 3.3 induced a hexagonal phase at the peptide-to-lipid ratio of 1/20 and, interestingly, cholesterol was not found in the hexagonal phase induced by aurein 3.3. The experimental results indicate that magainin 2 is more effective in disrupting lipid bilayers containing cholesterol than aurein 3.3.

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“…2). Phospholamban is a transmembrane peptide that regulates cardiac contractility and spans the lipid bilayer [15,16]. (KIGAKI) 3 is a positively charged peptide designed to have high antimicrobial activity and is known to strongly interact with the membrane surface [1,17].…”

Section: Resultsmentioning

confidence: 99%