Biology subjects

Eddy, M.

Publications and source records attributed to Eddy, M..

5 recordsLinked to original sources

bioRxiv2026

Spatial Organization of Lipids Drives GPCR Conformational Equilibria

Despite the widespread use of lipid nanodiscs for structure-function studies of membrane proteins, little is known about how lipids are spatially organized within nanodiscs and how this organization influences embedded proteins. The activity and conformational equilibria of the human A2A adenosine receptor, a class A G protein-coupled receptor, are highly sensitive to anionic lipids that directly interact with the receptor. We leverage this lipid-dependent sensitivity to probe the accessibility of anionic lipids across nanodiscs of varying sizes. We identify a threshold concentration of anionic lipids required to fully populate active receptor conformations that is higher for POPS than for POPG. Computational simulations reveal that POPS and POPG each form lipid clusters reducing the effective availability of anionic lipids to interact with the receptor. This effect is stronger for POPS and scales with increasing nanodisc size, correlating with experimental biophysical and biochemical measurements. Simulations further identify positively charged residues within the membrane scaffold protein that coordinate anionic lipid headgroups. Targeted protein engineering reduces the threshold concentration of anionic lipids required for receptor activation, supporting strategies to control lipid accessibility within nanodiscs. Because membrane scaffold proteins are derived from apolipoprotein A-I, similar lipid-protein interactions may also influence lipid organization within biological systems such as HDL particles.

biophysics↗

bioRxiv2024

The conformational equilibria of a human GPCR compared between lipid vesicles and aqueous solutions by integrative 19F-NMR

Endogenous phospholipids influence the conformational equilibria of G protein-coupled receptors, regulating their ability to bind drugs and form signaling complexes. However, most studies of GPCR-lipid interactions have been carried out in mixed micelles or lipid nanodiscs. Though useful, these membrane mimetics do not fully replicate the physical properties of native cellular membranes associated with large assemblies of lipids. We investigated the conformational equilibria of the human A2A adenosine receptor (A2AAR) in phospholipid vesicles using 19F solid-state magic angle spinning NMR (SSNMR). By applying an optimized sample preparation workflow and experimental conditions, we were able to obtain 19F-SSNMR spectra for both antagonist- and agonist-bound complexes with sensitivity and linewidths closely comparable to those achieved using solution NMR. This facilitated a direct comparison of the A2AAR conformational equilibria across detergent micelle, lipid nanodisc, and lipid vesicle preparations. While antagonist-bound A2AAR showed a similar conformational equilibria across all membrane and membrane mimetic systems, the conformational equilibria of agonist-bound A2AAR exhibited differences among different environments. This suggests that the conformational equilibria of GPCRs may be influenced not only by specific receptor-lipid interactions but also by the membrane properties found in larger lipid assemblies.

biophysics↗

bioRxiv2024

Visualizing the impact of disease-associated mutations on G protein-nucleotide interactions

Activation of G proteins stimulates ubiquitous intracellular signaling cascades essential for life processes. Under normal physiological conditions, nucleotide exchange is initiated upon the formation of complexes between a G protein and G protein-coupled receptor (GPCR), which facilitates exchange of bound GDP for GTP, subsequently dissociating the trimeric G protein into its G and G{beta}{gamma} subunits. However, single point mutations in G circumvent nucleotide exchange regulated by GPCR-G protein interactions, leading to either loss-of-function or constitutive gain-of-function. Mutations in several G subtypes are closely linked to the development of multiple diseases, including several intractable cancers. We leveraged an integrative spectroscopic and computational approach to investigate the mechanisms by which seven of the most frequently observed clinically-relevant mutations in the subunit of the stimulatory G protein result in functional changes. Variable temperature circular dichroism (CD) spectroscopy showed a bimodal distribution of thermal melting temperatures across all GS variants. Modeling from molecular dynamics (MD) simulations established a correlation between observed thermal melting temperatures and structural changes caused by the mutations. Concurrently, saturation-transfer difference NMR (STD- NMR) highlighted variations in the interactions of GS variants with bound nucleotides. MD simulations indicated that changes in local interactions within the nucleotide-binding pocket did not consistently align with global structural changes. This collective evidence suggests a multifaceted energy landscape, wherein each mutation may introduce distinct perturbations to the nucleotide-binding site and protein-protein interaction sites. Consequently, it underscores the importance of tailoring therapeutic strategies to address the unique challenges posed by individual mutations.

biophysics↗

bioRxiv2023

Anionic Phospholipids Control Mechanisms of GPCR-G Protein Recognition

G protein-coupled receptors (GPCRs) are embedded in phospholipids that strongly influence drug-stimulated signaling. Anionic lipids are particularly important for GPCR signaling complex formation, but a mechanism for this role is not understood. Using NMR spectroscopy, we visualized the impact of anionic lipids on the function-related conformational equilibria of the human A2A adenosine receptor (A2AAR) in bilayers containing defined mixtures of zwitterionic and anionic phospholipids. Anionic lipids primed the receptor to form complexes with G proteins through a conformational selection process. Without anionic lipids, signaling complex formation proceeded through a less favorable induced fit mechanism. In computational models, anionic lipids mimicked interactions between a G protein and positively charged residues in A2AAR at the receptor intracellular surface, stabilizing a pre-activated receptor conformation. Replacing these residues strikingly altered the receptor response to anionic lipids in experiments. High sequence conservation of the same residues among all GPCRs supports a general role for lipid-receptor charge complementarity in signaling.

biophysics↗

bioRxiv2020

The P600 ERP component as an index of rational error correction within a noisy-channel framework of human communication

Recent evidence suggests that language processing is well-adapted to noise in the input (e.g., speech errors, mishearing) and readily corrects the input via rational inference over possible intended sentences and probable noise corruptions. However, it remains unclear whether this inference takes the form of an offline re-analysis or a rapid, real-time correction to the representations of the input. We hypothesize that noise inferences happen online during processing and that well-studied ERP components may serve as a useful index of this process. In particular, a reduced N400 effect and increased P600 effect appear to accompany sentences where the probability that the message was corrupted by noise exceeds the probability that it was produced intentionally and perceived accurately. Indeed, semantic violations that are attributable to noise--for example, in "The storyteller could turn any incident into an amusing antidote", where the implausible word "antidote" is orthographically and phonologically close to the intended "anecdote"--elicit a reduced N400 effect and larger P600 effect. Further, the magnitude of this P600 effect is shown to relate to the probability that the comprehender will retrieve a plausible alternative. This work thus adds to the growing body of literature that suggests that many aspects of language processing are well-adapted to noise in the input and opens the door to electrophysiologic investigations of these processes

neuroscience↗