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Journal ArticleDOI

Magnetically oriented dodecylphosphocholine bicelles for solid-state NMR structure analysis.

01 May 2012-Biochimica et Biophysica Acta (Elsevier)-Vol. 1818, Iss: 5, pp 1142-1147
TL;DR: A mixture of 1,2-dimyristoyl-sn-glycero-3-phosphocholine with the short-chain detergent n-dodecylphosph Cocholine is introduced here as a new membrane-mimetic bicelle system for solid-state NMR structure analysis of membrane proteins in oriented samples.
About: This article is published in Biochimica et Biophysica Acta.The article was published on 2012-05-01 and is currently open access. It has received 21 citations till now. The article focuses on the topics: Model lipid bilayer.
Citations
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Journal ArticleDOI
TL;DR: This work worked with two major Aβ variants and handled them as if they were membrane proteins and found that the Aβ variant most strongly linked to AD assembled into stable Aβ oligomers that adopted a specific structure and incorporated into membranes as pores, a feature linked to neurotoxicity.
Abstract: The formation of amyloid-β peptide (Aβ) oligomers at the cellular membrane is considered to be a crucial process underlying neurotoxicity in Alzheimer’s disease (AD). Therefore, it is critical to characterize the oligomers that form within a membrane environment. To contribute to this characterization, we have applied strategies widely used to examine the structure of membrane proteins to study the two major Aβ variants, Aβ40 and Aβ42. Accordingly, various types of detergent micelles were extensively screened to identify one that preserved the properties of Aβ in lipid environments—namely the formation of oligomers that function as pores. Remarkably, under the optimized detergent micelle conditions, Aβ40 and Aβ42 showed different behavior. Aβ40 aggregated into amyloid fibrils, whereas Aβ42 assembled into oligomers that inserted into lipid bilayers as well-defined pores and adopted a specific structure with characteristics of a β-barrel arrangement that we named β-barrel pore-forming Aβ42 oligomers (βPFOsAβ42). Because Aβ42, relative to Aβ40, has a more prominent role in AD, the higher propensity of Aβ42 to form βPFOs constitutes an indication of their relevance in AD. Moreover, because βPFOsAβ42 adopt a specific structure, this property offers an unprecedented opportunity for testing a hypothesis regarding the involvement of βPFOs and, more generally, membrane-associated Aβ oligomers in AD.

214 citations


Cites background or methods from "Magnetically oriented dodecylphosph..."

  • ...β-(1-40) and β-(1-42) peptides of Alzheimer’s disease....

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  • ...Mandal PK, Pettegrew JW (2004) Alzheimer’s disease: Soluble oligomeric Abeta(1-40)...

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  • ...Lambert MP, et al. (1998) Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins....

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  • ...Shafrir Y, Durell S, Arispe N, Guy HR (2010) Models of membrane-bound Alzheimer’s Abeta peptide assemblies....

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  • ...However, the DMPC/DPC bicelle system had only previously established for the preparation of large bicelles useful for solid-state NMR measurements (26)....

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Journal ArticleDOI
TL;DR: An introduction to the properties of lipid bicelle phases with an emphasis on NMR experimental measurements is given and some of the most exciting recent applications of bicelles in the structural and dynamic studies of membrane proteins are discussed.

112 citations

Journal ArticleDOI
TL;DR: This work presents a strategy that creates small isotropic bicelles from IMPs co-translationally embedded in large nanodiscs using cell-free expression and validate the approach on the detergent-sensitive LspA, which finally allowed us to perform high-quality triple-resonance NMR experiments for structural studies.

24 citations


Additional excerpts

  • ...the phosphodiester group (Nolandt et al., 2012)....

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Journal ArticleDOI
TL;DR: The detailed topology found in this work agrees with the peptides forming the rim of nanodiscs in a double belt arrangement, and the tilt and helical pitch angles are well suited to cover the hydrophobic chain region of the bilayer when two peptide helices form a head-to-tail dimer.

20 citations

Journal ArticleDOI
TL;DR: It is demonstrated that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion, and it is validated that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane.
Abstract: Bacterial cell lysis during bacteriophage infection is timed by perfect orchestration between components of the holin-endolysin cassette. In bacteria, progressively accumulating holin in the inner membrane, retained in its inactive form by antiholin, is triggered into active hole formation, resulting in the canonical host cell lysis. However, the molecular mechanism of regulation and physical basis of pore formation in the mycobacterial cell membrane by D29 mycobacteriophage holin, particularly in the nonexistence of a known antiholin, is poorly understood. In this study, we report, for the first time, the use of fluorescence resonance transfer measurements to demonstrate that the first transmembrane domain (TM1) of D29 holin undergoes a helix ↔ β-hairpin conformational interconversion. We validate that this structural malleability is mediated by a centrally positioned proline and is responsible for controlled TM1 self-association in membrana, in the presence of a proton gradient across the lipid membrane. We demonstrate that TM1 is sufficient for bacterial growth inhibition. The biological effect of D29 holin structural alteration is presented as a holin self-regulatory mechanism, and its implications are discussed in the context of holin function.

19 citations

References
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Journal ArticleDOI
TL;DR: The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.
Abstract: Mixtures of long-chain and short-chain phosphatidylcholine (PC) were characterized by multinuclear (13C, 31P, 2H) solid-state nuclear magnetic resonance. This work complements and extends previous characterization of such mixtures by focusing on concentrated mixtures at temperatures above the gel to liquid crystalline phase transition temperature (Tm) of the long-chain PC component. Above Tm it was observed that highly oriented, bilayer-like assemblies could be formed of mixtures of dimyristoylphosphatidylcholine (DMPC) and dihexanoylphosphatidylcholine (DHPC) in molar ratios ranging from approximately 1:3.5 to 1:2 (DHPC:DMPC) over a considerable range of lipid concentrations (at least 3-40% w/v total lipid, for a 1:2.5 sample). Orientation was observed to occur only in an L alpha-like phase. The NMR data can be accounted for by a general model of the DHPC-DMPC aggregates in which DHPC can be found in two distinct populations (one highly ordered, one not). The averaged conformations of the glycerol backbone/headgroup regions of the long- and short-chain PC composing the assemblies were judged by solid-state 13C NMR to be similar to each other. The information gleaned about these mixtures and the quality of the oriented NMR spectra obtained suggest that DHPC-DMPC mixtures may prove to be useful as model membrane media in solid-state NMR studies of biomembranes.

451 citations

Journal ArticleDOI
TL;DR: The three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution is determined using heteronuclear NMR.
Abstract: We have determined the three-dimensional fold of the 19 kDa (177 residues) transmembrane domain of the outer membrane protein A of Escherichia coli in dodecylphosphocholine (DPC) micelles in solution using heteronuclear NMR. The structure consists of an eight-stranded beta-barrel connected by tight turns on the periplasmic side and larger mobile loops on the extracellular side. The solution structure of the barrel in DPC micelles is similar to that in n-octyltetraoxyethylene (C(8)E(4)) micelles determined by X-ray diffraction. Moreover, data from NMR dynamic experiments reveal a gradient of conformational flexibility in the structure that may contribute to the membrane channel function of this protein.

392 citations

Journal ArticleDOI
TL;DR: By solution NMR methods, the atomic resolution structure of an unphosphorylated PLN pentamer in dodecylphosphocholine micelles is determined, revealing a channel-forming architecture that could allow passage of small ions.
Abstract: Contraction and relaxation of heart muscle cells is regulated by cycling of calcium between cytoplasm and sarcoplasmic reticulum. Human phospholamban (PLN), expressed in the sarcoplasmic reticulum membrane as a 30-kDa homopentamer, controls cellular calcium levels by a mechanism that depends on its phosphorylation. Since PLN was discovered ≈30 years ago, extensive studies have aimed to explain how it influences calcium pumps and to determine whether it acts as an ion channel. We have determined by solution NMR methods the atomic resolution structure of an unphosphorylated PLN pentamer in dodecylphosphocholine micelles. The unusual bellflower-like assembly is held together by leucine/isoleucine zipper motifs along the membrane-spanning helices. The structure reveals a channel-forming architecture that could allow passage of small ions. The central pore gradually widens toward the cytoplasmic end as the transmembrane helices twist around each other and bend outward. The dynamic N-terminal amphipathic helices point away from the membrane, perhaps facilitating recognition and inhibition of the calcium pump.

312 citations

Journal ArticleDOI
TL;DR: Results indicate that bicelles may be uniquely and effectively employed as model membranes to facilitate NMR structural studies of many, but not all, membrane proteins.
Abstract: This paper describes a study undertaken to assess the possibility and practical consequences of reconstituting integral and peripheral membrane proteins into bilayered discoidal mixed micelles ("bicelles") composed of dimyristoylphosphatidylcholine and smaller amounts of either CHAPSO or short-chain phosphatidylcholine. The amphiphilic assemblies in these mixtures are uniquely suited for use in NMR structural studies because they can be magnetically oriented with experimentally-tunable system order. The first step of this study was to test about 15 membrane-associating polypeptides and proteins for their ability to interfere with magnetic orientation of the bicellar assemblies. A variety of results were obtained ranging from no perturbation to a complete disruption of orientation. Second, the suitability of bicelles as mimics of natural bilayers was tested by reconstituting diacylglycerol kinase, an integral membrane enzyme. The kinase was observed to be functional and completely stable for at least 24 h when incubated at 38 degrees C in bicelles. Third, the NMR spectra from a number of bicelle-reconstituted proteins were examined. In some cases, 13C NMR resonances from reconstituted proteins were extremely broad and asymmetric. In other cases, resonances from reconstituted proteins were moderately broad, but much less so than resonances from proteins reconstituted into multilayers oriented by mechanical methods. In the cases of two surface-associating proteins (cytochrome c and leucine enkephalin), oriented sample 13C NMR spectra of extremely high resolution were obtained. For these proteins it was also demonstrated that the experimentally variable order of the bicellar assemblies could be exploited to provide a means of screening for detergent-specific structural perturbations, for making spectral assignments, and for measuring chemical shift anisotropies and dipolar couplings. Taken as a whole, these results indicate that bicelles may be uniquely and effectively employed as model membranes to facilitate NMR structural studies of many, but not all, membrane proteins.

309 citations

Journal ArticleDOI
TL;DR: High-resolution structural detail, such as differences in tilt and rotational orientation along the helical axis leading to an assessment of helical coiling, can be obtained.

293 citations