The factors defining the correct folding and stability of integral membrane proteins are poorly understood. Folding of only a few select membrane proteins has been scrutinised, leaving considerable ...deficiencies in knowledge for large protein families, such as G protein coupled receptors (GPCRs). Complete reversible folding, which is problematic for any membrane protein, has eluded this dominant receptor family. Moreover, attempts to recover receptors from denatured states are inefficient, yielding at best 40-70% functional protein. We present a method for the reversible unfolding of an archetypal family member, the β1-adrenergic receptor, and attain 100% recovery of the folded, functional state, in terms of ligand binding, compared to receptor which has not been subject to any unfolding and retains its original, folded structure. We exploit refolding on a solid support, which could avoid unwanted interactions and aggregation that occur in bulk solution. We determine the changes in structure and function upon unfolding and refolding. Additionally, we employ a method that is relatively new to membrane protein folding; pulse proteolysis. Complete refolding of β1-adrenergic receptor occurs in n-decyl-β-D-maltoside (DM) micelles from a urea-denatured state, as shown by regain of its original helical structure, ligand binding and protein fluorescence. The successful refolding strategy on a solid support offers a defined method for the controlled refolding and recovery of functional GPCRs and other membrane proteins that suffer from instability and irreversible denaturation once isolated from their native membranes.
A deficiency of thiamine (vitamin B1), an essential cofactor for enzymes involved in metabolic processes, can be caused by the enzyme thiaminase. Thiaminase in food stocks has been linked to ...morbidity and mortality due to thiamine depletion in many ecologically and economically important species. Thiaminase activity has been detected in certain bacteria, plants, and fish species, including carp. The invasive silver carp (Hypophthalmichthys molitrix) presents an enormous burden to ecosystems throughout the Mississippi River watershed. Its large biomass and nutritional content offer an attractive possibility as a food source for humans, wild animals, or pets. Additionally, harvesting this fish could alleviate some of the effects of this species on waterways. However, the presence of thiaminase would detract from its value for dietary consumption. Here we confirm the presence of thiaminase in several tissues from silver carp, most notably the viscera, and systematically examine the effects of microwaving, baking, dehydrating, and freeze-drying on thiaminase activity. Certain temperatures and durations of baking and microwaving reduced thiaminase activity to undetectable levels. However, caution should be taken when carp tissue is concentrated by processes without sufficient heat treatment, such as freeze-drying or dehydration, which results in concentration, but not inactivation of the enzyme. The effects of such treatments on the ease of extracting proteins, including thiaminase, and the impact on data interpretation using the 4-nitrothiophenol (4-NTP) thiaminase assay were considered.
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•Thiaminase I is an enzyme that breaks down the essential vitamin thiamine (vitamin B1).•Thiaminase I activity was found in invasive silver carp (Hypophthalmichthys molitrix).•We tested thiaminase I activity before and after various common food processing steps.•Processes without sufficient heat treatment dehydrate and concentrate enzyme activity.•Certain microwaving or baking times and temperatures effectively reduced the activity.
Silver nanoparticles (Ag NPs) can be found in myriad consumer products, medical equipment/supplies, and public spaces. However, questions remain regarding the risks associated with Ag NP exposure. As ...part of a consortium-based effort to better understand these nanomaterials, this study examined how Ag NPs with varying sizes and coatings affect pulmonary responses at different time-points. Four types of Ag NPs were tested: 20 nm (C20) and 110 nm (C110) citrate-stabilized NPs, and 20 nm (P20) and 110 nm (P110) PVP-stabilized NPs. Male, Sprague Dawley rats were intratracheally instilled with Ag NPs (0, 0.1, 0.5, or 1.0 mg/kg bodyweight BW), and bronchoalveolar lavage fluid (BALF) and lung tissues were obtained at 1, 7, and 21 days post-exposure for analysis of BAL cells and histopathology. All Ag NP types produced significantly elevated polymorphonuclear cells (PMNs) in BALF on Days 1, 7, and/or 21 at the 0.5 and/or 1.0 mg/kg BW dose(s). Histology of animals exposed to 1.0 mg/kg BW Ag NPs showed patchy, focal, centriacinar inflammation for all time-points; though neutrophils, macrophages, and/or monocytes were also found in the airway submucosa and perivascular regions at Days 1 and 7. Confocal microscopy of ethidium homodimer-stained lungs at Day 1 showed dead/dying cells at branch points along the main airway. By Day 21, only animals exposed to the high dose of C110 or P110 exhibited significant BALF neutrophilia and marked cellular debris in alveolar airspaces. Findings suggest that 110 nm Ag NPs may produce lasting effects past Day 21 post instillation.
4-Methylimidazole (4MEI) is a contaminant in food and consumer products. Pulmonary toxicity and carcinogenicity following chronic dietary exposures to 4MEI is a regulatory concern based on previous ...rodent studies. This study examined acute pulmonary toxicity in B6C3F1 mice from 6 h to 5 days after oral gavage with a single dose of 150 mg/kg 4MEI, a double dose delivered 6 h apart, or vehicle controls. Oral gavage of 150 mg/kg naphthalene, a prototypical Club cell toxicant, was used as a positive control. Intrapulmonary conducting airway cytotoxicity was assessed in fixed-pressure inflated lungs using qualitative histopathology scoring, quantitative morphometric measurement of vacuolated and exfoliating epithelial cells, and immunohistochemistry. 4MEI treatment did not change markers of cytotoxicity including the mass of vacuolated epithelium, the thickness of the epithelium, or the distributions of epithelial proteins: secretoglobin 1A1, proliferating cell nuclear antigen, calcitonin gene-related peptide, and myeloperoxidase. 4MEI and vehicle controls caused slight cytotoxicity with rare vacuolization of the epithelium relative to the severe bronchiolar epithelial cell toxicity found in the naphthalene exposed mice at terminal bronchioles, intrapulmonary airways, or airway bifurcations. In summary, 4MEI caused minimal airway epithelial toxicity without characteristic Club Cell toxicity when compared to naphthalene, a canonical Club Cell toxicant.
•No mouse lung cell damage by high resolution histopathology after 2 high 4MEI doses.•Mouse lung cell damage after naphthalene gavage mirrored known intraperitoneal toxicity.•4MEI lacked characteristic Club Cell toxicity, especially compared to naphthalene.
Overexpression of mammalian membrane proteins in mammalian cells is an effective strategy to produce sufficient protein for biophysical analyses and structural studies, because the cells generally ...express proteins in a correctly folded state. However, obtaining high levels of expression suitable for protein purification on a milligram scale can be challenging. As membrane protein overexpression often has a negative impact on cell viability, it is usual to make stable cell lines where the protein of interest is expressed from an inducible promoter. Here we describe a methodology for optimizing the inducible production of any membrane protein fused to GFP through the isolation of clonal cell lines. Flow cytometry is used to sort uninduced cells and the most fluorescent 5 % of the cell population are used to make clonal cell lines.
Increasing silver nanoparticle (AgNP) use in sprays, consumer products, and medical devices has raised concerns about potential health effects. While previous studies have investigated AgNPs, most ...were limited to a single particle size or surface coating. In this study, we investigated the effect of size, surface coating, and dose on the persistence of silver in the lung following exposure to AgNP. Adult male rats were intratracheally instilled with four different AgNPs: 20 or 110 nm in size and coated with either citrate or polyvinylpyrrolidone (PVP) at 0.5 or 1.0 mg/kg doses. Silver retention was assessed in the lung at 1, 7, and 21 d post exposure. ICP-MS quantification demonstrated that citrate-coated AgNPs persisted in the lung to 21 d with retention greater than 90%, while PVP-coated AgNP had less than 30% retention. Localization of silver in lung tissue at 1 d post exposure demonstrated decreased silver in proximal airways exposed to 110 nm particles compared with 20 nm AgNPs. In terminal bronchioles 1 d post exposure, silver was localized to surface epithelium but was more prominent in the basement membrane at 7 d. Silver positive macrophages in bronchoalveolar lavage fluid decreased more quickly after exposure to particles coated with PVP. We conclude that PVP-coated AgNPs had less retention in the lung tissue over time and larger particles were more rapidly cleared from large airways than smaller particles. The 20 nm citrate particles showed the greatest effect, increasing lung macrophages even 21 d after exposure, and resulted in the greatest silver retention in lung tissue.
The structure of the rhodopsin/mini-G
o
complex reveals new insights on G protein selectivity.
Selective coupling of G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptors ...(GPCRs) to specific Gα-protein subtypes is critical to transform extracellular signals, carried by natural ligands and clinical drugs, into cellular responses. At the center of this transduction event lies the formation of a signaling complex between the receptor and G protein. We report the crystal structure of light-sensitive GPCR rhodopsin bound to an engineered mini-G
o
protein. The conformation of the receptor is identical to all previous structures of active rhodopsin, including the complex with arrestin. Thus, rhodopsin seems to adopt predominantly one thermodynamically stable active conformation, effectively acting like a “structural switch,” allowing for maximum efficiency in the visual system. Furthermore, our analysis of the well-defined GPCR–G protein interface suggests that the precise position of the carboxyl-terminal “hook-like” element of the G protein (its four last residues) relative to the TM7/helix 8 (H8) joint of the receptor is a significant determinant in selective G protein activation.
Integral membrane proteins do not fare well when extracted from biological membranes and are unstable or lose activity in detergents commonly used for structure and function investigations. We show ...that phospholipid bicelles provide a valuable means of preserving alpha-helical membrane proteins
in vitro by supplying a soluble lipid bilayer fragment. Both 1,2-dimyristoyl-
sn-glycero-3-phosphocholine (DMPC)/3-(cholamidopropyl)dimethyl-ammonio-1-propane sulfonate (Chaps) and DMPC/
l-α-1,2-dihexanoyl-
sn-glycero-3-phosphocholine (DHPC) bicelles dramatically increase the stability of the mammalian vision receptor rhodopsin as well as its apoprotein, opsin. Opsin is particularly unstable in detergent solution but can be directly purified into DMPC/Chaps. We show that opsin can also be directly purified in DMPC/DHPC bicelles to give correctly folded functional opsin, as shown by the ability to regenerate rhodopsin to ∼
70% yield. These well-characterised DMPC/DHPC bicelles enable us to probe the influence of bicelle properties on opsin stability. These bicelles are thought to provide DMPC bilayer fragments with most DHPC capping the bilayer edge, giving a soluble bilayer disc. Opsin stability is shown to be modulated by the
q value, the ratio of DMPC to DHPC, which reflects changes in the bicelle size and, thus, proportion of DMPC bilayer present. The observed changes in stability also correlate with loss of opsin secondary structure as determined by synchrotron far-UV circular dichroism spectroscopy; the most stable bicelle results in the least helix loss. The inclusion of Chaps rather than DHPC in the DMPC/Chaps bicelles, however, imparts the greatest stability. This suggests that it is not just the DMPC bilayer fragment in the bicelles that stabilises the protein, but that Chaps provides additional stability either through direct interaction with the protein or by altering the DMPC/Chaps bilayer properties within the bicelle. The significant stability enhancements and preservation of secondary structure reported here in bicelles are pertinent to other membrane proteins, notably G-protein-coupled receptors, which are unstable in detergent solution.
The key to determining crystal structures of membrane protein complexes is the quality of the sample prior to crystallization. In particular, the choice of detergent is critical, because it affects ...both the stability and monodispersity of the complex. We recently determined the crystal structure of an active state of bovine rhodopsin coupled to an engineered G protein, mini-Go, at 3.1 Å resolution. Here, we detail the procedure for optimizing the preparation of the rhodopsin-mini-Go complex. Dark-state rhodopsin was prepared in classical and neopentyl glycol (NPG) detergents, followed by complex formation with mini-Go under light exposure. The stability of the rhodopsin was assessed by ultraviolet-visible (UV-VIS) spectroscopy, which monitors the reconstitution into rhodopsin of the light-sensitive ligand, 9-cis retinal. Automated size-exclusion chromatography (SEC) was used to characterize the monodispersity of rhodopsin and the rhodopsin-mini-Go complex. SDS-polyacrylamide electrophoresis (SDS-PAGE) confirmed the formation of the complex by identifying a 1:1 molar ratio between rhodopsin and mini-Go after staining the gel with Coomassie blue. After cross-validating all this analytical data, we eliminated unsuitable detergents and continued with the best candidate detergent for large-scale preparation and crystallization. An additional problem arose from the heterogeneity of N-glycosylation. Heterologously-expressed rhodopsin was observed on SDS-PAGE to have two different N-glycosylated populations, which would probably have hindered crystallogenesis. Therefore, different deglycosylation enzymes were tested, and endoglycosidase F1 (EndoF1) produced rhodopsin with a single species of N-glycosylation. With this strategic pipeline for characterizing protein quality, preparation of the rhodopsin-mini-Go complex was optimized to deliver the crystal structure. This was only the third crystal structure of a GPCR-G protein signaling complex. This approach can also be generalized for other membrane proteins and their complexes to facilitate sample preparation and structure determination.
For the first time, protein microcrystallography has been performed with a focused synchrotron‐radiation beam of 1 µm using a goniometer with a sub‐micrometre sphere of confusion. The crystal ...structure of xylanase II has been determined with a flux density of about 3 × 1010 photons s−1 µm−2 at the sample. Two sets of diffraction images collected from different sized crystals were shown to comprise data of good quality, which allowed a 1.5 Å resolution xylanase II structure to be obtained. The main conclusion of this experiment is that a high‐resolution diffraction pattern can be obtained from 20 µm3 crystal volume, corresponding to about 2 × 108 unit cells. Despite the high irradiation dose in this case, it was possible to obtain an excellent high‐resolution map and it could be concluded from the individual atomic B‐factor patterns that there was no evidence of significant radiation damage. The photoelectron escape from a narrow diffraction channel is a possible reason for reduced radiation damage as indicated by Monte Carlo simulations. These results open many new opportunities in scanning protein microcrystallography and make random data collection from microcrystals a real possibility, therefore enabling structures to be solved from much smaller crystals than previously anticipated as long as the crystallites are well ordered.
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