Wayne L. Hubbell

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) is an established tool for exploring protein structure and dynamics. Although nitroxide side chains attached to a single cysteine via a disulfide linkage are commonly employed in SDSL-EPR, their internal flexibility complicates applications to monitor slow internal motions in proteins and to structure determination by distance mapping. Moreover, the labile disulfide linkage prohibits the use of reducing agents often needed for protein stability. To enable the application of SDSL-EPR to the measurement of slow internal dynamics, new spin labels with hindered internal motion are desired. Here, we introduce a highly ordered nitroxide side chain, designated R9, attached at a single cysteine residue via a non-reducible thioether linkage. The reaction to introduce R9 is highly selective for solvent-exposed cysteine residues. Structures of R9 at two helical sites in T4 Lysozyme were determined by X-ray crystallography and the mobility in helical sequences was characterized by EPR spectral lineshape analysis, Saturation Transfer EPR, and Saturation Recovery EPR. In addition, interspin distance measurements between pairs of R9 residues are reported. Collectively, all data indicate that R9 will be useful for monitoring slow internal structural fluctuations, and applications to distance mapping via dipolar spectroscopy and relaxation enhancement methods are anticipated.

The μ-opioid receptor (μOR) is an important target for pain management and the molecular understanding of drug action will facilitate the development of better therapeutics. Here we show, using double electron-electron resonance (DEER) and single-molecule fluorescence resonance energy transfer (smFRET), how ligand-specific conformational changes of the μOR translate into a broad range of intrinsic efficacies at the transducer level. We identify several cytoplasmic receptor conformations interconverting on different timescales, including a pre-activated receptor conformation which is capable of G protein binding, and a fully activated conformation which dramatically lowers GDP affinity within the ternary complex. Interaction of β-arrestin-1 with the μOR core binding site appears less specific and occurs with much lower affinity than binding of G protein G _i . One-Sentence Summary: Ligand-dependent conformational dynamics of the μ-opioid receptor determine downstream signaling efficacy.

Defining the denatured state ensemble (DSE) and disordered proteins is essential to understanding folding, chaperone action, degradation, and translocation. As compared with water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we measure the DSE of the helical membrane protein GlpG of Escherichia coli ( E. coli ) in native-like lipid bilayers. The DSE was obtained using our steric trapping method, which couples denaturation of doubly biotinylated GlpG to binding of two streptavidin molecules. The helices and loops are probed using limited proteolysis and mass spectrometry, while the dimensions are determined using our paramagnetic biotin derivative and double electron–electron resonance spectroscopy. These data, along with our Upside simulations, identify the DSE as being highly dynamic, involving the topology changes and unfolding of some of the transmembrane (TM) helices. The DSE is expanded relative to the native state but only to 15 to 75% of the fully expanded condition. The degree of expansion depends on the local protein packing and the lipid composition. E. coli ’s lipid bilayer promotes the association of TM helices in the DSE and, probably in general, facilitates interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes.

G protein-coupled receptors (GPCRs) represent a large class of transmembrane helical proteins which are involved in numerous physiological signaling pathways and therefore represent crucial pharmacological targets. GPCR function and the action of therapeutic molecules are defined by only a few parameters, including receptor basal activity, ligand affinity, intrinsic efficacy and signal bias. These parameters are encoded in characteristic receptor conformations existing in equilibrium and their populations, which are thus of paramount interest for the understanding of receptor (mal-)functions and rational design of improved therapeutics. To this end, the combination of site-directed spin labeling and EPR spectroscopy, in particular double electron–electron resonance (DEER), is exceedingly valuable as it has access to sub-Angstrom spatial resolution and provides a detailed picture of the number and populations of conformations in equilibrium. This review gives an overview of existing DEER studies on GPCRs with a focus on the delineation of structure/function frameworks, highlighting recent developments in data analysis and visualization. We introduce “conformational efficacy” as a parameter to describe ligand-specific shifts in the conformational equilibrium, taking into account the loose coupling between receptor segments observed for different GPCRs using DEER.

Defining the denatured state ensemble (DSE) and intrinsically disordered proteins is essential to understanding protein folding, chaperone action, degradation, translocation and cell signaling. While a majority of studies have focused on water-soluble proteins, the DSE of membrane proteins is much less characterized. Here, we reconstituted the DSE of a helical-bundle membrane protein GlpG of Escherichia coli in native lipid bilayers and measured the DSE’s conformation and compactness. The DSE was obtained using steric trapping, which couples spontaneous denaturation of a doubly biotinylated GlpG to binding of two bulky monovalent streptavidin molecules. Using limited proteolysis and mass spectrometry, we mapped the flexible regions in the DSE. Using our paramagnetic biotin derivative and double electron-electron resonance spectroscopy, we determined the dimensions of the DSE. Finally, we employed our Upside model for molecular dynamics simulations to generate the DSE including the collapsed and fully expanded states in a bilayer. We find that the DSE is highly dynamic involving the topology changes of transmembrane segments and their unfolding. The DSE is expanded relative to the native state, but only to 55–90% of the fully expanded condition. The degree of expansion depends on the chemical potential with regards to local packing and the lipid composition. Our result suggests that the E. coli ’s native lipid bilayer promotes the association of helices in the DSE of membrane proteins and, probably in general, facilitating interhelical interactions. This tendency may be the outcome of a general lipophobic effect of proteins within the cell membranes. Significance: Here, we delineate the conformation of the denatured state ensemble (DSE) of a membrane protein confined in a native lipid bilayer and assay whether the bilayer permits full expansion or nonspecific collapse of the DSE. Using the intramembrane protease GlpG as a model, we find that the denatured state is a dynamic ensemble involving topological changes and local unfolding of transmembrane segments. The bilayer tends to contract the DSE relative to the fully lipid-solvated, expanded conformations while the degree of compactness is determined by the balance between protein-lipid, lipid-lipid and protein-protein interactions. These findings provide new insights into the lipid bilayer as a solvent that mediates folding, chaperone action, turnover and protein-protein interactions in cell membranes.

Significance: The molecular origin of basal activity in G protein coupled receptors and its suppression by inverse agonists are poorly understood. Here we employ pressure-resolved DEER spectroscopy to explore the structural origin of basal activity and the mechanism of inverse agonism in the β ₂ adrenergic receptor. Collectively, the results support a conformational selection model wherein basal activity is due to a preexisting equilibrium between inactive and active conformations in the unliganded receptor, and inverse agonists stabilize the inactive conformation. Furthermore, the results illustrate the importance of rare states in molecular mechanisms of protein function and the capability of pressure-resolved DEER for revealing structural features of sparsely populated conformations of membrane proteins and for determining their thermodynamic properties.

Voltage sensing domains (VSDs) couple changes in transmembrane electrical potential to conformational changes that regulate ion conductance through a central channel. Positively charged amino acids inside each sensor cooperatively respond to changes in voltage. Our previous structure of a TPC1 channel captured the first example of a resting-state VSD in an intact ion channel. To generate an activated state VSD in the same channel we removed the luminal inhibitory Ca ²⁺ -binding site (Ca _i ²⁺ ), that shifts voltage-dependent opening to more negative voltage and activation at 0 mV. Cryo-EM reveals two coexisting structures of the VSD, an intermediate state 1 that partially closes access to the cytoplasmic side, but remains occluded on the luminal side and an intermediate activated state 2 in which the cytoplasmic solvent access to the gating charges closes, while luminal access partially opens. Activation can be thought of as moving a hydrophobic insulating region of the VSD from the external side, to an alternate grouping on the internal side. This effectively moves the gating charges from the inside potential to that of the outside. Activation also requires binding of Ca ²⁺ to a cytoplasmic site (Ca _a ²⁺ ). An X-ray structure with Ca _a ²⁺ removed and a near-atomic resolution cryo-EM structure with Ca _i ²⁺ removed define how dramatic conformational changes in the cytoplasmic domains may communicate with the VSD during activation. Together four structures provide a basis for understanding the voltage dependent transition from resting to activated state, the tuning of VSD by thermodynamic stability, and this channel’s requirement of cytoplasmic Ca ²⁺ -ions for activation.

Significance: Activation of G protein-coupled receptors (GPCRs) initiates conformational shifts that trigger interaction with a specific G-protein subtype from a structurally homologous set. A major unsolved problem is the mechanism by which this selectivity is achieved. Structures of GPCR–G protein complexes so far fail to reveal the origin of selectivity because they all involve one G-protein subtype (G_s). In this work, we report a structural model of the activated GPCR rhodopsin in complex with another G-protein subtype (G_i) derived from intermolecular distance mapping with DEER-EPR and refinement with modeling. Comparison of the model with structures of complexes involving G_sreveals distinct GPCR–G protein-binding modes, the differences of which suggest key features of the structural selectivity filter.

Significance: Cellular acidification occurs in response to many cellular events, including ischemia and apoptosis. The voltage-dependent anion channel (VDAC) is the most abundant protein in the outer mitochondrial membrane and has been implicated in mitochondrial regulation, cell death, and assembling protein complexes with Bcl2 family of apoptotic regulating proteins. During apoptosis, VDAC has demonstrated rapid oligomerization followed by cytochrome c release into the cytoplasm. We report a new dimeric model of VDAC induced by minor acidification with a pKa of 7.4, which is regulated by the protonation state of transmembrane residue E73. This study links a specific dimer formation with cellular acidification, which may be a salient feature of the molecular mechanism(s) involved in mitochondrial regulation.