Biology subjects

Hubbell, W. L.

Publications and source records attributed to Hubbell, W. L..

4 recordsLinked to original sources

bioRxiv2026

Conformational Basis of Functionally Selective Allosteric Modulation of the Angiotensin II type 1 Receptor by Small Molecules

Blockade of signaling through the angiotensin II type 1 receptor (AT1R), a prototypical G protein-coupled receptor (GPCR), by angiotensin receptor blockers (ARBs) is a major therapeutic approach to treating a wide variety of cardiovascular and renal diseases1. Like most GPCRs, the AT1R signals through two transducers, G proteins and {beta}-arrestins2,3. Previous reports have described {beta}-arrestin-biased peptide orthosteric agonists for the AT1R with potential therapeutic advantages over currently available unbiased ARBs4-6. Here we report the DNA- encoded library screening-guided isolation and pharmacological characterization of the first small molecule AT1R allosteric ligands. We use cryo-electron microscopy, double electron- electron resonance spectroscopy, molecular dynamics simulations, and targeted mutagenesis to determine their binding sites, binding modes and conformational mechanisms driving their unique and divergent modulatory effects on G protein and {beta}-arrestin pathways. Our findings uncover new mechanisms for precisely controlling the dynamic behavior of the AT1R with implications for drug development targeting this pathophysiologically important receptor family.

biochemistry↗

bioRxiv2024

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-s using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of s-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components ([~]3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

biophysics↗

bioRxiv2023

Conformational dynamics of the μ-opioid receptor determine ligand intrinsic efficacy

The -opioid receptor (OR) 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 OR 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 {beta}-arrestin-1 with the OR core binding site appears less specific and occurs with much lower affinity than binding of G protein Gi. One-Sentence SummaryLigand-dependent conformational dynamics of the -opioid receptor determine downstream signaling efficacy.

biochemistry↗

bioRxiv2021

Lipid Bilayer Induces Contraction of the Denatured State Ensemble of a Helical-Bundle Membrane Protein

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 DSEs 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. colis 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. SignificanceHere, 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.

biophysics↗