Rohan D. A. Alvares

Role of Detergents in Conformational Exchange of a G Protein-coupled Receptor

Background: Membrane protein functional dynamics are sensitive to the detergent host. Results: Three functional states of the β2-adrenoreceptor (β2AR) are identified in maltose-neopentyl glycol, whereas all states exchange rapidly in dodecyl maltoside. Conclusion: β2AR 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. The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles, the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is 4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange between distinct functional states associated with the G protein-coupled receptor.

Using [1‐13C]lactic acid for hyperpolarized 13C MR cardiac studies

Hyperpolarized [1‐13C]lactate in solution may be a clinically relevant and safe substrate for real time MR investigations of key metabolic pathways. The potential of using hyperpolarized [1‐13C]lactate for magnetic resonance studies of cardiac metabolism in vivo was explored.

Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway.

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.

Quantitative Detection of PEGylated Biomacromolecules in Biological Fluids by NMR

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.

Direct quantitative 13C-filtered 1H magnetic resonance imaging of PEGylated biomacromolecules in vivo

1H MRI is an established diagnostic method that generally relies on detection of water. Imaging specific macromolecules is normally accomplished only indirectly through the use of paramagnetic tags, which alter the water signal in their vicinity. We demonstrate a new approach in which macromolecular constituents, such as proteins and drug delivery systems, are observed directly and quantitatively in vivo using 1H MRI of 13C‐labeled poly(ethylene glycol) (13C‐PEG) tags.

A scale to measure MRI contrast agent sensitivity

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.

Using [1-(13) C]lactic acid for hyperpolarized (13) C MR cardiac studies.

Hyperpolarized [1‐13C]lactate in solution may be a clinically relevant and safe substrate for real time MR investigations of key metabolic pathways. The potential of using hyperpolarized [1‐13C]lactate for magnetic resonance studies of cardiac metabolism in vivo was explored.

Using [1-13C]lactic acid for hyperpolarized13C MR cardiac studies: Lactic Acid for13C MR Cardiac Studies

Hyperpolarized [1‐13C]lactate in solution may be a clinically relevant and safe substrate for real time MR investigations of key metabolic pathways. The potential of using hyperpolarized [1‐13C]lactate for magnetic resonance studies of cardiac metabolism in vivo was explored.