Background: Membrane protein functional dynamics are sensitive to the detergent host. Results: Three functional states of the  2 -adrenoreceptor ( 2 AR) are identified in maltose-neopentyl glycol, whereas all states exchange rapidly in dodecyl maltoside. Conclusion:  2 AR converts between inactive and active states on a time scale that depends on the detergent off-rate. Significance: G protein-coupled receptor functional dynamics are understood by considering topology changes and corresponding rearrangements of associated detergents.
Poly(acrylic acid) consisting of 25 monomer units (PAA 25 ) was used to stabilize nanoparticle aggregates (NPAs) consisting of either NaGdF 4 or 50/50 mixtures of GdF 3 and CeF 3 . The resulting polymer-stabilized nanoparticle aggregates (NPAs) were developed and tested for their application as contrast agents for magnetic resonance imaging (MRI) and computed tomography (CT). The PAA 25 -stabilized NPAs exhibit low polydispersity and are colloidally stable at concentrations of 40 mg/mL, while their sizes can be be controlled by choosing a specific ratio of Gd 3þ to Ce 3þ . Scanning transmission electron microscopy (STEM) reveals that NaGdF 4 NPAs possess an average diameter of 400 nm. High-resolution STEM and powder X-ray diffraction (XRD) both show that these NPAs consist of a stable aggregate of smaller NPs, whose diameters are 20-22 nm. PAA 25stabilized NPAs consisting of a 50/50 mixture of GdF 3 and CeF 3 possess an average diameter of 70 nm, while the fundamental unit size is estimated to be 10-12 nm in diameter. The PAA 25 -stabilized GdF 3 /CeF 3 NPAs possess mass relaxivities of 40 ( 2 and 30 ( 2 s -1 (mg/mL) -1 at 1.5 T and 3.0 T, respectively. Their effectiveness as contrast agents for CT X-ray imaging at various X-ray energies was also tested and compared to that of equivalent mass concentrations of Gd 3þ -diethylene triamine pentaacetic acid (Gd 3þ -DTPA) and iopromide. Gd-based NPAs exhibit superior CT contrast to equal-mass concentrations of either iopromide or Gd 3þ -DTPA below 30 keV and above 50 keV. Finally, PAA 25 was functionalized by folic acid to explore targeted imaging. Confocal microscopy revealed that, by functionalizing the PAA 25 -stabilized NaGdF 4 :Tb 3þ NPAs with ∼0.8 folates per polymer, binding and endocytosis occurred in SK-BR-3 human breast cancer cells. The utility of the PAA 25 -stabilized GdF 3 /CeF 3 NPAs for MRI is demonstrated in rat perfusion MRI experiments, where T 1 -weighted MRI images of equivalent concentrations of either Gd 3þ -DTPA or the above NPAs are directly compared. The high relaxivities provide an opportunity to conduct perfusion MRI experiments with significantly lower concentrations than those needed for current commercial agents.
This study demonstrated that the use of neat [1-(13) C]lactic acid as the DNP sample is a potential alternative to [1-(13) C]pyruvic acid for cardiac hyperpolarized (13) C MR studies. Hyperpolarized [1-(13) C]lactate may enable noninvasive assessment of cardiac PDH flux in cardiac patients in the near future.
Magnetic resonance imaging (MRI) provides superior resolution of anatomical features and the best soft tissue contrast, and is one of the predominant imaging modalities. With this technique, contrast agents are often used to aid discrimination by enhancing specific features. Over the years, a rich diversity of such agents has evolved and with that, so has a need to systematically sort contrast agents based on their efficiency, which directly determines sensitivity. Herein, we present a scale to rank MRI contrast agents. The scale is based on analytically determining the minimum detectable concentration of a contrast agent, and employing a ratiometric approach to standardize contrast efficiency to a benchmark contrast agent. We demonstrate the approach using several model contrast agents and compare the relative sensitivity of these agents for the first time. As the first universal metric of contrast agent sensitivity, this scale will be vital to easily assessing contrast agent efficiency and thus important to promoting use of some of the elegant and diverse contrast agents in research and clinical practice.
Membrane proteins constitute a significant fraction of the proteome and are important drug targets. While the transmembrane (TM) segments of these proteins are primarily composed of hydrophobic residues, the inclusion of polar residues-either naturally occurring or as a consequence of a disease-related mutation-places a significant folding burden in this environment, potentially impacting bilayer insertion and/or association of neighboring TM helices. Here we investigate the role of an anionic detergent, sodium dodecylsulfate (SDS), and a zwitterionic detergent, dodecylphosphocholine (DPC), in the folding process, and the effects induced by a single polar substitution, on structure and topology of model α-helical TM segments. The peptides, represented by KK-YAAAIAAIAWAXAAIAAAIAA-KKK-NH(2), where X is I or N, are designed with high aqueous solubilities, through poly-lysine tags. Circular dichroism (CD) and NMR were used to monitor peptide secondary structure and diffusional mobility of both peptide and the detergent hosts. For both peptides, SDS binding commenced at a concentration below its CMC, due to Coulombic attraction of anionic SDS to cationic Lys residues. Increasing SDS binding correlated with increasing peptide helicity. Pulsed field gradient (PFG) NMR diffusion measurements revealed that the Asn-containing peptide bound four fewer detergent molecules, corresponding to ca. 20% less SDS than bound by the Ile peptide. Conversely, zwitterionic DPC binding to either peptide was not observed until the DPC concentration approached its CMC. Our findings confirm quantitatively that a single polar residue within a TM segment may have a significant influence on its local membrane environment.
The accumulation, biodistribution, and clearance profiles of therapeutic agents are key factors relevant to their efficacy. Determining these properties constitutes an ongoing experimental challenge. Many such therapeutics, including small molecules, peptides, proteins, tissue scaffolds, and drug delivery vehicles, are conjugated to poly(ethylene glycol) (PEG) as this improves their bioavailability and in vivo stability. We demonstrate here that (1)H NMR spectroscopy can be used to quantify PEGylated species in complex biological fluids directly, rapidly, and with minimal sample preparation. PEG bears a large number of spectroscopically equivalent protons exhibiting a narrow NMR line width while resonating at a (1)H NMR frequency distinct from most other biochemical signals. We demonstrate that PEG provides a robust signal allowing detection of concentrations as low as 10 μg/mL in blood. This PEG detection limit is lowered by another order of magnitude when background proton signals are minimized using (13)C-enriched PEG in combination with a double quantum filter to remove (1)H signals from non-(13)C-labeled species. Quantitative detection of PEG via these methods is shown in pig blood and goat serum as examples of complex biological fluids. More practically, we quantify the blood clearance of (13)C-PEG and PEGylated-BSA (bovine serum albumin) following their intravenous injection in live rats. Given the relative insensitivity of line width to PEG size, we anticipate that the biodistribution and clearance profiles of virtually any PEGylated biomacromolecule from biological fluid samples can be routinely measured by (1)H NMR without any filtering or treatment steps.
Bulk thermodynamic and volumetric parameters (ΔGmic°, ΔHmic°, ΔSmic°, ΔCp,mic°, ΔVmic°, and Δκmic°) associated with the monomer–micelle equilibrium, were directly determined for a variety of common detergents [sodium n-dodecyl sulfate (SDS), n-dodecyl phosphocholine (DPC), n-dodecyl-β-d-maltoside (DDM), and 7-cyclohexyl-1-heptyl phosphocholine (CyF)] via 1H NMR spectroscopy. For each temperature and pressure point, the critical micelle concentration (cmc) was obtained from a single 1H NMR spectrum at a single intermediate concentration by referencing the observed chemical shift to those of pure monomer and pure micellar phases. This permitted rapid measurements of the cmc over a range of temperatures and pressures. In all cases, micelle formation was strongly entropically favored, while enthalpy changes were all positive, with the exception of SDS, which exhibited a modestly negative enthalpy of micellization. Heat capacity changes were also characteristically negative, while partial molar volume changes were uniformly positive, as expected for an aggregation process dictated by hydrophobic effects. Isothermal compressibility changes were found to be consistent with previous measurements using other techniques. Thermodynamic measurements were also related to spectroscopic studies of topology and micelle structure. For example, paramagnetic effects resulting from the addition of dioxygen provided microscopic topological details concerning the hydrophobicity gradient along the detergent chains within their respective micelles as detected by 1H NMR. In a second example, combined 13C and 1H NMR chemical shift changes arising from application of high pressure, or upon micellization, of CyF provided site-specific details regarding micelle topology. In this fashion, bulk thermodynamics could be related to microscopic topological details within the detergent micelle.
PEGylation is widely employed to enhance the performance of a multitude of macromolecular therapeutics and drug delivery systems, and C-filtered H MRI of C-PEG thus offers the possibility of imaging and quantitating their distribution in living systems in real time. Magn Reson Med 77:1553-1561, 2017. © 2016 International Society for Magnetic Resonance in Medicine.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.