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Proteine in Zeitlupe: 19F ‐ NMR ‐ Studien deuten darauf hin, dass Übergänge zwischen aktiven und inaktiven Zuständen des humanen β2 ‐ adrenergen Rezeptors erhebliche Strukturumwandlungen erfordern (siehe Bild). Dies zeigt sich an der Enthalpiedifferenz ΔHo≈40 kJ mol−1 und einer langsamen Umwandlungsgeschwindigkeit mit kex≪10 s−1. [ABSTRACT FROM AUTHOR]

Mikrospulen ‐ NMR ‐ Messungen wurden durchgeführt, um die Zusammensetzung von detergenshaltigen β2 ‐ Adrenozeptor ‐ Lösungen (β2AR) zu bestimmen und die hydrodynamischen Eigenschaften der β2AR ‐ Detergensmicellen (siehe Bild) zu untersuchen. Es werden Standards zur reproduzierbaren Herstellung von Lösungen G ‐ Protein ‐ gekoppelter Rezeptoren für Kristallisationsversuche und Flüssig ‐ NMR ‐ Untersuchungen etabliert. [ABSTRACT FROM AUTHOR]

Aqueous solutions of the detergent Fos-10 (n-decylphosphocholine)without and with addition of the integral membrane protein (IMP) OmpX(outer membrane protein X) have been characterized using pulsed fieldgradient-stimulated echo (PFG-STE) NMR experiments for measurementsof translational diffusion coefficients. Effective diffusion coefficientsfor Fos-10 micelles in the absence of OmpX were obtained by observationof NMR signals from 10-bromodecan-1-ol that had been inserted intothe micelles, and in the presence of OmpX by NMR observation of theprotein. It is thus shown that solutions of Fos-10-reconstituted OmpXcan be quantitatively described as a mixture of Fos-10 monomers, uniformFos-10 micelles, and uniform OmpX-containing Fos-10 micelles, withFos-10 monomers in fast exchange between the pools of these threespecies. This result establishes an avenue for efficient determinationof the effective translational diffusion coefficients of IMP-containingdetergent micelles based on observation of the intense detergent NMRsignals, which is also applicable with unlabeled IMPs. This monitoringof the species present in a given IMP solution contributes to improvedguidelines for rational selection of detergent and buffer conditionsin structural studies of integral membrane proteins. [ABSTRACT FROM AUTHOR]

The article looks at an experiment to observe the cytoplasmic region of the G protein-coupled receptor (GPCR) type beta-adrenergic receptors (β2AR) bound with extracellular ligands with fluorine nuclear magnetic resonance (19F-NMR) imaging. Analysis of the binding with various ligands indicated that the conformational equilibria shifted selectively towards either helices Vl or Vll of the beta-adrenergic receptors depending on the ligand. The results indicates that β2ARs demonstrate biased signalling toward β-arrestin- or G protein-specific agonists due to the structural plasticity of its conformation.

The rate limiting step in biophysical characterization of membrane proteins is often the availability of suitable amounts of protein material. It was therefore of interest to demonstrate that microcoil nuclear magnetic resonance (NMR) technology can be used to screen microscale quantities of membrane proteins for proper folding in samples destined for structural studies. Micoscale NMR was then used to screen a series of newly designed zwitterionic phosphocholine detergents for their ability to reconstitute membrane proteins, using the previously well characterized β-barrel E. coli outer membrane protein OmpX as a test case. Fold screening was thus achieved with microgram amounts of uniformly ²H,15N-labeld OmpX and affordable amounts of the detergents, and prescreening with SDS-gel electrophoresis ensured efficient selection of the targets for NMR studies. A systematic approach to optimize the phosphocholine motif for membrane protein refolding led to the identification of two new detergents, 138-Fos and 179-Fos, that yield 2D [15N, ¹H]-TROSY correlation NMR spectra of natively folded reconstituted OmpX. [ABSTRACT FROM AUTHOR]

Summary Signaling across cellular membranes, the 826 human G protein-coupled receptors (GPCRs) govern a wide range of vital physiological processes, making GPCRs prominent drug targets. X-ray crystallography provided GPCR molecular architectures, which also revealed the need for additional structural dynamics data to support drug development. Here, nuclear magnetic resonance (NMR) spectroscopy with the wild-type-like A 2A adenosine receptor (A 2A AR) in solution provides a comprehensive characterization of signaling-related structural dynamics. All six tryptophan indole and eight glycine backbone 15 N– 1 H NMR signals in A 2A AR were individually assigned. These NMR probes provided insight into the role of Asp52 2.50 as an allosteric link between the orthosteric drug binding site and the intracellular signaling surface, revealing strong interactions with the toggle switch Trp 246 6.48 , and delineated the structural response to variable efficacy of bound drugs across A 2A AR. The present data support GPCR signaling based on dynamic interactions between two semi-independent subdomains connected by an allosteric switch at Asp52 2.50 . [ABSTRACT FROM AUTHOR]

Microcoil NMR measurements were performed to determine the final composition of solutions of the β(2)-adrenergic receptor (β(2)AR) reconstituted with a detergent and to study the hydrodynamic properties of the detergent micelles containing β(2)AR. Standards are established for the reproducible preparation of G-protein-coupled receptor solutions for crystallization trials and solution NMR studies., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)