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There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into β-DDMB yielded activities that were 40% higher than those of DAGK purified into β-DDM. β-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. β-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.
PURPOSE - Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome.
METHODS - Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-β-cyclodextrin was quantified by iTRAQ analysis.
RESULTS - A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains.
CONCLUSIONS - These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.
To facilitate fine-scale phenotyping of whole specimens, we describe here a set of tissue fixation-embedding, detergent-clearing and staining protocols that can be used to transform excised organs and whole organisms into optically transparent samples within 1-2 weeks without compromising their cellular architecture or endogenous fluorescence. PACT (passive CLARITY technique) and PARS (perfusion-assisted agent release in situ) use tissue-hydrogel hybrids to stabilize tissue biomolecules during selective lipid extraction, resulting in enhanced clearing efficiency and sample integrity. Furthermore, the macromolecule permeability of PACT- and PARS-processed tissue hybrids supports the diffusion of immunolabels throughout intact tissue, whereas RIMS (refractive index matching solution) grants high-resolution imaging at depth by further reducing light scattering in cleared and uncleared samples alike. These methods are adaptable to difficult-to-image tissues, such as bone (PACT-deCAL), and to magnified single-cell visualization (ePACT). Together, these protocols and solutions enable phenotyping of subcellular components and tracing cellular connectivity in intact biological networks.
Bilayered detergent-lipid assemblies known as bicelles have been widely used as model membranes in structural biological studies and are being explored for wider applications, including pharmaceutical use. Most studies to date have involved the use of concentrated bicelle mixtures, such that little is known about the capacity of bicellar mixtures to be diluted without unwanted transitions to nonisotropic phases. Here, different detergent/lipid mixtures have been explored, leading to the identification of two different families of bicelles for which it is possible to lower the total amphiphile (detergent + lipid) concentration to <1% (w/v) while retaining isotropic assemblies. These include a novel family of bicelles based on mixtures of 6-cyclohexyl-1-hexylphosphocholine (Cyclofos-6) and the lipid dimyristoylphosphatidylcholine (DMPC). Bicelles formed by these mixtures can be diluted to <0.5% and also have attractive biochemical properties. However, a caveat of our results is that the diffusion coefficients measured for the lipid component of the different bicelles tested were seen to be dependent on sample history, even though all samples were optically transparent. This suggests that the phase behavior of bicelles at low lipid-to-detergent ratios may be more complex than previously appreciated.
G protein coupled receptors (GPCRs) can be activated by various extracellular stimuli, including hormones, peptides, odorants, neurotransmitters, nucleotides, or light. After activation, receptors interact with heterotrimeric G proteins and catalyze GDP release from the Gα subunit, the rate limiting step in G protein activation, to form a high affinity nucleotide-free GPCR-G protein complex. In vivo, subsequent GTP binding reduces affinity of the Gα protein for the activated receptor. In this study, we investigated the biochemical and structural characteristics of the prototypical GPCR, rhodopsin, and its signaling partner, transducin (G(t)), in bicelles to better understand the effects of membrane composition on high affinity complex formation, stability, and receptor mediated nucleotide release. Our results demonstrate that the high-affinity complex (rhodopsin-G(t)(empty)) forms more readily and has dramatically increased stability when rhodopsin is integrated into bicelles of a defined composition. We increased the half-life of functional complex to 1 week in the presence of negatively charged phospholipids. These data suggest that a membrane-like structure is an important contributor to the formation and stability of functional receptor-G protein complexes and can extend the range of studies that investigate properties of these complexes.
Sodium dodecylbenzenesulfonate is one of a group of salts of alkylbenzene sulfonates used in cosmetics as surfactant-cleansing agents. Sodium dodecylbenzenesulfonate is soluble in water and partially soluble in alcohol, with dermal absorption dependent on pH. Dodecylbenzenesulfonate salts are not toxic in single-dose oral and dermal animal tests, and no systemic toxicities were observed in repeat-dose dermal animal studies. In dermal animal studies, no evidence of reproductive or developmental toxicity was reported. At 15% concentrations, sodium dodecylbenzenesulfonate was severely irritating to rabbit skin. The Cosmetic Ingredient Review Expert Panel concluded that the irritant properties of these ingredients are similar to those of other detergents, with severity dependent on concentration and pH. Products containing these ingredients should be formulated to ensure that the irritancy potential is minimized.
Matrix-Assisted Laser Desorption/Ionization (MALDI) Imaging Mass Spectrometry (IMS) is a molecular technology that allows simultaneous investigation of the content and spatial distribution of molecules within tissue. In this work, we examine different classes of detergents, the anionic sodium dodecyl sulfate (SDS), the nonionic detergents Triton X-100, Tween 20 and Tween 80, and the zwitterionic 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) for use in MALDI IMS of analytes above m/z 4000. These detergents were found to be compatible with MALDI MS and did not cause signal suppression relative to non-detergent applications and did not produce interfering background signals. In general, these detergents enhanced signal acquisition within the mass range m/z 4-40 000. Adding detergents into the matrix was comparable with the separate application of detergent and matrix. Evaluation of spectra collected from organ-specific regions of a whole mouse pup section showed that different detergents perform optimally with different organs, indicating that detergent selection should be optimized on the specific tissue for maximum gain. These data show the utility of detergents towards enhancement of protein signals for on-tissue MALDI IMS analysis.
Copyright © 2010 John Wiley & Sons, Ltd.
Cholesterol and its hemisuccinate and sulfate derivatives are widely used in studies of purified membrane proteins but are difficult to solubilize in aqueous solution, even in the presence of detergent micelles. Other cholesterol derivatives do not form conventional micelles and lead to viscous solutions. To address these problems, a cholesterol-based detergent, CHOBIMALT, has been synthesized and characterized. At concentrations above 3−4 μM, CHOBIMALT forms micelles without the need for elevated temperatures or sonic disruption. Diffusion and fluorescence measurements indicated that CHOBIMALT micelles are large (210±30 kDa). The ability to solubilize a functional membrane protein was explored using a G-protein coupled receptor, the human kappa opioid receptor type 1 (hKOR1). While CHOBIMALT alone was not found to be effective as a surfactant for membrane extraction, when added to classical detergent micelles CHOBIMALT was observed to dramatically enhance the thermal stability of solubilized hKOR1.
Plakophilins (Pkp-1, -2, and -3) comprise a family of armadillo repeat-containing proteins first identified as desmosomal plaque components, in which they link desmoplakin to the desmosomal cadherins. In addition to their role in desmosomal cell-cell adhesion, Pkps also localize to the nucleus, where they perform unknown functions. Of the three Pkps, Pkp-1 is most readily detected in the nucleus, where it is localized to the nucleoplasm. Pkp chimeras containing the Pkp-1 head domain and Pkp-3 armadillo repeat domain were localized to the nucleus in A431 cells, whereas Pkp chimeras containing the Pkp-3 head domain and Pkp-1 armadillo repeat domain localized to the desmosome and the cytosol. DNAse I digestion of chromatin in cultured cells results in loss of nuclear Pkp-1, suggesting that Pkp-1 associates specifically with nuclear components. In addition, in vitro assays revealed that the amino-terminal head domains of Pkps-1 and -2 were sufficient to bind single-stranded DNA. Induction of DNA damage induced a partial redistribution of Pkp-1 protein to the nucleolus, and depletion of Pkp-1 resulted in increased survival in response to DNA damage. These data suggest that in addition to mediating desmosome assembly, the nuclear pool of Pkp can influence cell survival by interactions with DNA.
G protein-coupled receptors (GPCRs) catalyze nucleotide release in heterotrimeric G proteins, the slow step in G protein activation. G i/o family proteins are permanently, cotranslationally myristoylated at the extreme amino terminus. While myristoylation of the amino terminus has long been known to aid in anchoring G i proteins to the membrane, the role of myristoylation with regard to interaction with activated receptors is not known. Previous studies have characterized activation-dependent changes in the amino terminus of Galpha proteins in solution [Medkova, M. (2002) Biochemistry 41, 9963-9972; Preininger, A. M. (2003) Biochemistry 42, 7931-7941], but changes in the environment of specific residues within the Galpha i1 amino terminus during receptor-mediated G i activation have not been reported. Using site-specific fluorescence labeling of individual residues along a stretch of the Galpha il amino terminus, we found specific changes in the environment of these residues upon interaction with the activated receptor and following GTPgammaS binding. These changes map to a distinct surface of the amino-terminal helix opposite the Gbetagamma binding interface. The receptor-dependent fluorescence changes are consistent with a myristoylated amino terminus in the proximity of the membrane and/or receptor. Myristoylation affects both the rate and intensity of receptor activation-dependent changes detected at several residues along the amino terminus (with no significant effect on the rate of receptor-mediated GTPgammaS binding). This work demonstrates that the myristoylated amino terminus of Galpha il proteins undergoes receptor-mediated changes during the dynamic process of G protein signaling.