G protein-coupled receptors can adopt many different conformational states, each of them exhibiting different restraints towards downstream signaling pathways. One promising strategy to identify and ...quantify this conformational landscape is to introduce a cysteine at a receptor site sensitive to different states and label this cysteine with a probe for detection. Here, the application of NMR of hyperpolarized 129Xe for the detection of the conformational states of human neuropeptide Y2 receptor is introduced. The xenon trapping cage molecule cryptophane-A attached to a cysteine in extracellular loop 2 of the receptor facilitates chemical exchange saturation transfer experiments without and in the presence of native ligand neuropeptide Y. High-quality spectra indicative of structural states of the receptor–cage conjugate were obtained. Specifically, five signals could be assigned to the conjugate in the apo form. After the addition of NPY, one additional signal and subtle modifications in the persisting signals could be detected. The correlation of the spectroscopic signals and structural states was achieved with molecular dynamics simulations, suggesting frequent contact between the xenon trapping cage and the receptor surface but a preferred interaction with the bound ligand.
We present a protocol to evaluate the utility of detergents for purification and delipidation of E. coli membrane proteins. We determine the critical aggregation concentration of detergents. ...Furthermore, we compare the ability of detergents to extract membrane proteins and to maintain protein-lipid interactions during purification. The protocol describes steps for isolating and delipidating membrane proteins from E. coli membranes by extraction and affinity purification using detergents. The protocol does not enable an absolute quantification of purification outcomes.
For complete details on the use and execution of this protocol, please refer to Urner et al.1
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•Methodic screening of detergents for the purification of membrane proteins•Understanding the aggregation behavior of a detergent•Extraction and affinity purification of membrane proteins•Estimation of relative delipidating properties of detergents by native MS
Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.
We present a protocol to evaluate the utility of detergents for purification and delipidation of E. coli membrane proteins. We determine the critical aggregation concentration of detergents. Furthermore, we compare the ability of detergents to extract membrane proteins and to maintain protein-lipid interactions during purification. The protocol describes steps for isolating and delipidating membrane proteins from E. coli membranes by extraction and affinity purification using detergents. The protocol does not enable an absolute quantification of purification outcomes.
The demand for responsive dyes in optical imaging is high to achieve a better signal-to-noise ratio and, more specifically, to visualize acidic compartments of the endocytic pathway. Herein, we ...present a new synthetic route, with a step-by-step synthesis of water-soluble pH-sensitive cyanine dyes exhibiting pK a values in the region of physiological pH, as confirmed by absorption and fluorescence spectra. Moreover, modification of pK a values was achieved by two different substitution patterns, creating tunable pH-sensitive dyes. We demonstrated the functionality of the pH-sensitive dyes and their suitability as contrast agents for cellular uptake studies by preparing dye-labeled cetuximab and transferrin conjugates. Sulfonated head chains increased water solubility and prevented the formation of dimers, even in the context of dye-labeled bioconjugates. Confocal microscopy images of living cells revealed their pH-responsiveness, as specific fluorescence signal enhancements were observed in acidic compartments of the endocytic pathway (endosomes and lysosomes), although the background signal was low in a pH-neutral environment. Using mixtures of conjugates labeled with either a pH-sensitive or non-pH-sensitive dye for the uptake studies, we could follow the receptor binding and distinguish it from the endocytic uptake process of the conjugates in a simultaneous manner. Moreover, we used flow cytometry to quantify the fluorescence and observed a 3-fold signal enhancement for the pH-sensitive dye conjugates over a period of 3 h.
Since therapeutic agents target specific compartments inside the cells, their efficiency depends on their intracellular release from drug delivery systems (DDS). However, control over the ...intracellular release of therapeutic agents is a challenging issue and can only be achieved by governing their interactions with the DDS. In this work, polyglycerol amine- and polyglycerol sulfate-functionalized graphene sheets as positively and negatively charged 2D nanomaterials with 150 nm lateral size were used to deliver and control the release of doxorubicin (DOX) inside cells. A pH-sensitive dye was conjugated onto the surfaces of graphene sheets and used as an antenna to obtain specific signals from the acidic cell compartments. It was found that both positively and negatively charged graphene sheets undergo similar acidification processes after cellular uptake. Nevertheless, the intracellular drug release of these DOX-loaded nanomaterials was distinctly different. As an overall effect of the π-π stacking and electrostatic interactions, the release of DOX from the positively charged graphene sheets was much faster than that from their analogs with a negative surface charge. Therefore, therapeutic efficiency in the first case was much higher than that in the latter. Based on our findings, the intracellular release of drugs from the surfaces of graphene sheets can be finely tuned by manipulating their functionalities, which is of great importance in the designing of the future graphene-based nanomedicines.
Detergent chemistry enables applications in the world today while harming safe operating spaces that humanity needs for survival. Aim of this review is to support a holistic thought process in the ...design of detergent chemistry. We harness the planetary boundary concept as a framework for literature survey to identify progresses and knowledge gaps in context with detergent chemistry and five planetary boundaries that are currently transgressed, i.e., climate, freshwater, land system, novel entities, biosphere integrity. Our survey unveils the status of three critical challenges to be addressed in the years to come, including (i) the implementation of a holistically, climate‐friendly detergent industry; (ii) the alignment of materialistic and social aspects in creating technical solutions by means of sustainable chemistry; (iii) the development of detergents that serve the purpose of applications but do not harm the biosphere in their role as novel entities. Specifically, medically relevant case reports revealed that even the most sophisticated detergent design cannot sufficiently accelerate drug discovery to outperform the antibiotic resistance development that detergents simultaneously promote as novel entities. Safe operating spaces that humanity needs for its survival may be secured by directing future efforts beyond sustainable chemistry, resource efficiency, and net zero emission targets.
It's detergent o'clock and the role of detergent chemistry in transgressing Earth's resources is reviewed to support future research that secures safe operating spaces that humanity needs for its survival.
The cover feature image illustrates the chemical diversity of the detergentome (entirety of all detergents). Detergents – no matter what color or shape – enable many applications in the world today. ...The description of the detergentome provided by Virginia Wycisk and co‐workers will facilitate project planning and the synthesis of detergents for challenging future applications. Cover design by Leonhard H. Urner. More information can be found in the Review by Wycisk, Wagner, and Urner.
The synthesis of a new amphiphilic 5,5′,6,6′-tetrachlorobenzimidacarbocyanine dye derivative with −(CH2)2–(CF2)5–CF3 chains attached to the nitrogen atoms in the 1,1′-position, CF8O3, is reported. ...Depending on the dye concentration and the addition of MeOH, CF8O3 forms J- and H-aggregates in aqueous solutions. The aggregation behavior was investigated using steady-state absorption, linear dichroism, and fluorescence spectroscopy, as well as by cryogenic transmission electron microscopy (cryo-TEM). The J-band of the MeOH-free solution is monomer-like, rather broad, and less red-shifted with respect to the monomer absorption, indicating weak excitonic coupling and disorder effects. Cryo-TEM reveals a diversity of supramolecular structures, wherein linear and branched cylindrical micelles dominate. It is concluded that the high stiffness of fluoroalkyl chains does not allow the chains to splay and completely fill up the hydrophobic gap between opposing chromophores. This destabilizes the bilayers and favors the micellar structure motifs instead. The aggregates appearing at 30% MeOH show a split absorption spectrum consisting of a broad blue-shifted H-band and an accompanying sharp red-shifted J-band with perpendicular polarizations. These HJ-type aggregates are also composed of micellar fibers, but these bundle into rope-like strands. For 10% MeOH, a narrow bilayered tube is the dominating morphology. The observed MeOH dependence of aggregation reveals a clear cosolvent effect.
Here, we use cryo soft X-ray tomography (cryo-SXT), which delivers 3D ultrastructural volumes of intact cells without chemical fixation or staining, to gain insight about nanoparticle uptake for ...nanomedicine. We initially used dendritic polyglycerol sulfate (dPGS) with potential diagnostic and therapeutic applications in inflammation. Although dPGS-coated gold nanoparticle (dPGS-AuNP) uptake followed a conventional endocytic/degradative pathway in human lung epithelial cell lines (A549), with cryo-SXT, we detected ∼5% of dPGS-AuNPs in the cytoplasm, a level undetectable by confocal light microscopy. We also observed ∼5% of dPGS-AuNPs in a rarely identified subcellular site, namely, lipid droplets, which are important for cellular energy metabolism. Finally, we also found substantial changes in the quantity of cytoplasmic organelles upon dPGS-AuNP uptake over the 1–6 h incubation period; the number of small vesicles and mitochondria significantly increased, and the number of multivesicular bodies and the number and volume of lipid droplets significantly decreased. Although nearly all organelle numbers at 6 h were still significantly different from controls, most appeared to be returning to normal levels. To test for generality, we also examined cells after uptake of gold nanoparticles coated with a different agent, polyethylenimine (PEI), used for nucleic acid delivery. PEI nanoparticles did not enter lipid droplets, but they induced similar, albeit less pronounced, changes in the quantity of cytoplasmic organelles. We confirmed these changes in organelle quantities for both nanoparticle coatings by confocal fluorescence microscopy. We suggest this cytoplasmic remodeling could reflect a more common cellular response to coated gold nanoparticle uptake.
A switchable solvatochromic fluorescent dyad can be used to map ordering of lipids in vesicle membranes at a resolution better than the diffraction limit. Combining a Nile Red fluorophore with a ...photochromic spironaphthoxazine quencher allows the fluorescence to be controlled using visible light, via photoswitching and FRET quenching. Synthetic lipid vesicles of varying composition were imaged with an average 2.5‐fold resolution enhancement, compared to the confocal images. Ratiometric detection was used to probe the membrane polarity, and domains of different lipid ordering were distinguished within the same membrane.
A photoswitchable solvatochromic dye has been synthesized, comprising a Nile Red fluorophore and a spironaphthoxazine photoswitch. Visible light super‐resolution RESOLFT microscopy permits ordered and disordered membrane domains to be distinguished at resolution below the diffraction limit.
Ionic detergents enable applications and cause harm in biospheres due to cell toxicity. The utility of covalent combinations between ionic and non-ionic detergent headgroups in modulating cell ...toxicity remains speculative due to the yet rarely explored synthesis. We close this gap and establish the modular synthesis of ionic/non-ionic hybrid detergents. We restructure a combinatorial methallyl dichloride one-pot coupling into a two-step coupling, which reduces by-products, improves product yields, and enables the gram-scale preparation of asymmetric, cationic/non-ionic and anionic/non-ionic hybrid detergents. Our modular synthesis delivers new modalities for the design of ionic detergents, including an unprecedented scaling of properties that determine applications, such as charge, critical micelle concentration (cmc), solubilizing properties, hard water tolerance, and cell compatibility. We uncover that shielding the charge in ionic headgroups can switch the detergent species that is toxic to cells from monomers to mixtures of monomers and micellar assemblies. Establishing the chemistry of ionic/non-ionic hybrid detergents provides a missing evolutionary link in the structural comparison of ionic and non-ionic detergents, enables an easy synthesis access to yet unexplored chemical spaces of asymmetric hybrid materials, and delivers new modalities for designing the toxicity of supramolecular nanomaterials.
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