Allosteric nanobodies reveal the dynamic range and diverse mechanisms of G-protein-coupled receptor activation.
Nature 535(7612):448 (2016)
G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and ac...
Activation of the A2A adenosine G-protein-coupled receptor by conformational selection.
Nature 533(7602):265 (2016)
Conformational selection and induced fit are two prevailing mechanisms to explain the molecular basis for ligand-based activation of receptors. G-protein-coupled receptors are the largest class of cell surface receptors and are important drug targets. A molecular understanding of their activatio...
Quantitative Detection of PEGylated Biomacromolecules in Biological Fluids by NMR.
Analytical chemistry 88(7):3730 (2016)
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 dru...
Structural Insights into the Dynamic Process of β2-Adrenergic Receptor Signaling.
Cell 161(5):1101 (2015)
G-protein-coupled receptors (GPCRs) transduce signals from the extracellular environment to intracellular proteins. To gain structural insight into the regulation of receptor cytoplasmic conformations by extracellular ligands during signaling, we examine the structural dynamics of the cytoplasmi...
A comparison of chemical shift sensitivity of trifluoromethyl tags: optimizing resolution in ¹⁹F NMR studies of proteins.
Journal of Biomolecular NMR 62(1):97 (2015)
The elucidation of distinct protein conformers or states by fluorine ((19)F) NMR requires fluorinated moieties whose chemical shifts are most sensitive to subtle changes in the local dielectric and magnetic shielding environment. In this study we evaluate the effective chemical shift dispersion ...
Nuts and Bolts of CF3 and CH 3 NMR Toward the Understanding of Conformational Exchange of GPCRs.
Methods in Molecular Biology 1335:39 (2015)
With the advent of efficient protein expression and functional purification protocols, it is now possible to reconstitute many G protein-coupled receptors (GPCRs) in detergent micelles at concentrations of 25 μM or more. Such concentrations are sufficient for studies of conformational states and...
Conformational selection and functional dynamics of calmodulin: a (19)F nuclear magnetic resonance study.
Biochemistry (Washington) 53(36):5727 (2014)
Calcium-bound calmodulin (CaM-4Ca(2+)) is innately promiscuous with regard to its protein interaction network within the cell. A key facet of the interaction process involves conformational selection. In the absence of a binding peptide, CaM-4Ca(2+) adopts an equilibrium between a native state (...
New pipelines for novel allosteric GPCR modulators.
Biophysical Journal 107(2):287 (2014)
Temperature and pressure based NMR studies of detergent micelle phase equilibria.
Journal of Physical Chemistry B 118(21):5698 (2014)
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-maltosid...
¹⁹F NMR studies of a desolvated near-native protein folding intermediate.
Biochemistry (Washington) 52(34):5780 (2013)
Although many proteins are recognized to undergo folding via an intermediate, the microscopic nature of folding intermediates is less understood. In this study, ¹⁹F NMR and near-UV circular dichroism (CD) are used to characterize a transition to a thermal folding intermediate of calmodulin, a wa...
Dynamic equilibria between monomeric and oligomeric misfolded states of the mammalian prion protein measured by 19F NMR.
Journal of the American Chemical Society 135(28):10533 (2013)
The assembly of misfolded proteins is a critical step in the pathogenesis of amyloid and prion diseases, although the molecular mechanisms underlying this phenomenon are not completely understood. Here, we use (19)F NMR spectroscopy to examine the thermodynamic driving forces surrounding formati...
The role of ligands on the equilibria between functional states of a G protein-coupled receptor.
Journal of the American Chemical Society 135(25):9465 (2013)
G protein-coupled receptors exhibit a wide variety of signaling behaviors in response to different ligands. When a small label was incorporated on the cytosolic interface of transmembrane helix 6 (Cys-265), (19)F NMR spectra of the β2 adrenergic receptor (β2AR) reconstituted in maltose/neopentyl...
Effects of a polar amino acid substitution on helix formation and aggregate size along the detergent-induced peptide folding pathway.
Biochimica et Biophysica Acta 1828(2):373 (2013)
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-...
The dynamic process of β(2)-adrenergic receptor activation.
Cell 152(3):532 (2013)
G-protein-coupled receptors (GPCRs) can modulate diverse signaling pathways, often in a ligand-specific manner. The full range of functionally relevant GPCR conformations is poorly understood. Here, we use NMR spectroscopy to characterize the conformational dynamics of the transmembrane core of ...
Effect of juxtamembrane tryptophans on the immersion depth of Synaptobrevin, an integral vesicle membrane protein.
Biochimica et Biophysica Acta 1818(12):2994 (2012)
Proper positioning of membrane proteins in the host membrane is often critical to successful protein function. While hydrophobic considerations play a dominant role in determining the topology of a protein in the membrane, amphiphilic residues, such as tryptophan, may 'anchor' the protein near t...
Role of detergents in conformational exchange of a G protein-coupled receptor.
Journal of Biological Chemistry 287(43):36305 (2012)
The G protein-coupled β(2)-adrenoreceptor (β(2)AR) signals through the heterotrimeric G proteins G(s) and G(i) and β-arrestin. As such, the energy landscape of β(2)AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β(2)AR with a trifluoromethyl probe, (19)F NMR was us...
Lysine methylation strategies for characterizing protein conformations by NMR.
Journal of Biomolecular NMR 54(2):199 (2012)
In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR s...
Quantitative analysis of the effects of photoswitchable distance constraints on the structure of a globular protein.
Biochemistry (Washington) 51(32):6421 (2012)
Photoswitchable distance constraints in the form of photoisomerizable chemical cross-links offer a general approach to the design of reversibly photocontrolled proteins. To apply these effectively, however, one must have guidelines for the choice of cross-linker structure and cross-linker attach...
Current applications of 19F NMR to studies of protein structure and dynamics.
Progress in Nuclear Magnetic Resonance Spectros... 62:1 (2012)
► 19F molecular tags and labeling protocols for 19F NMR studies of proteins are reviewed and contrasted. ► 19F NMR biosynthetic labeling strategies are presented. ► Experimental challenges (loss of function through labeling, line broadening, assignment ambiguities)...
Topology and immersion depth of an integral membrane protein by paramagnetic rates from dissolved oxygen.
Journal of Biomolecular NMR 51(1-2):173 (2011)
In studies of membrane proteins, knowledge of protein topology can provide useful insight into both structure and function. In this work, we present a solution NMR method for the measurement the tilt angle and average immersion depth of alpha helices in membrane proteins, from analysis of the pa...