Robert Scott Prosser

Large poly(ethylene) glycol (PEG) chains are often conjugated to proteins or biomolecules to inhibit proteolytic degradation, mask immunogenic response, reduce clearance rates, and improve biodistribution of therapeutics, vaccines, drug delivery systems, and gene therapy formulations. The PEG macromolecular chain can also be used as a noninvasive reporter to track biologics in vivo by magnetic resonance spectroscopy (MRS). Rapid internal dynamics of PEG render the transverse ¹ H spin relaxation time to be comparable to water (~0.5 s) and amenable to imaging through traditional pulsed field gradient techniques. While water signal grossly exceeds that of PEG it is possible to filter ¹ H MRS signal of PEGylated conjugates through one of two ways—(1) stimulated echo acquisition mode (STEAM) MRS, which leverages huge differences in the diffusion of water versus PEGylated constructs, and (2) ¹³ C‐edited ¹ H MRS of fully ¹³ C‐enriched PEGylated constructs. Here, we compare both approaches. A 15 kDa ¹³ C‐enriched PEG chain was prepared alone, conjugated to bovine serum‐albumin (BSA), and incorporated into a 52‐nm‐diameter PEG‐poly(lactic acid) (PLA) nanoparticle. These three PEG constructs were then separately monitored in real time by ¹³ C‐edited ¹ H MRS, after introducing them into rat animal models intravenously. A ¹³ C‐editing scheme was employed to monitor ¹ H MRS PEG signal in the vasculature via a radiofrequency coil placed around the tail. An observed two‐component decay of the PEG signal is attributed to perfusion and early equilibration (alpha phase) and slow clearance (beta phase). ¹³ C‐PEG alone, ¹³ C‐PEG‐BSA, and ¹³ C‐PEG–PLA nanoparticles exhibited half‐lives of 38.6 min, 23.4 h, and 11.9 h, respectively. The relatively rapid clearance rates associated with the PEG–PLA nanoparticles is expected to arise from enzymatic degradation of the PLA chain. Using STEAM‐based editing schemes, we then evaluated sensitivity and water suppression in diffusion‐edited ¹ H MRS for ( ¹² C)‐PEGylated BSA contrasting 2‐, 20‐, and 40‐kDa PEG chains, in imaging phantom samples. Larger molecular weight PEG chains (i.e., 40 kDa) proved far superior to smaller PEG chain reporters due to reduced inhomogeneities and longer T ₂ , upon employing either a ¹³ C‐HQMC filter or a STEAM‐based diffusion filter.

The human adenosine A _2A receptor (A _2A R) is a prototypical member of the class A family of G Protein–Coupled Receptors (GPCRs), which are engaged by over one-third of FDA-approved drugs. Here, we used ¹⁹ F NMR to simultaneously evaluate functional states from the perspective of a CF ₃ -tag on transmembrane helix-6 (TM6) and all 5-fluorotryptophan reporters. Whereas spectra from the TM6 tag reveal a dynamic conformational ensemble, the tryptophan spectra exhibit more discrete ligand-dependent states. These ligand-dependent signatures provide insights into microswitches including a universal toggle switch which interfaces with a sodium binding pocket. Over 560 GPCRs share this toggle switch — sodium pocket cluster, implying a fundamental role in activation. Computational rigidity-theory reveals tryptophan reporters reside along prominent allosteric activation pathways, underscoring their roles in mechanical signal propagation and corroborating NMR observations. Among them, W246 ^6.48 , associated with the toggle switch, plays a critical role in regulation of allosteric networks spanning the orthosteric pocket and extending via tryptophan-rich pathways through the receptor to the A _2A R-Gβ interface and the nucleotide pocket in G _s α. While higher sodium concentrations (~100 mM) predictably stabilize the inactive conformation of the receptor, lower concentrations (below 40 mM) greatly enhanced the presence of the activation ensemble and in particular, the precoupled state, suggesting that the release of sodium from the conserved pocket—either through basal conditions for the apo receptor, or upon binding of the agonist—enables sampling of a precoupled state, ultimately needed to initiate activation and coupling.

Many G protein-coupled receptors (GPCRs) exhibit constitutive activity through spontaneous conformational transitions that facilitate G protein coupling and downstream signaling in the absence of ligand binding. This baseline activity, which is critical for cellular homeostasis, can be influenced by receptor conformational ensembles and plasma membrane organization. We used live-cell signaling assays, single-particle tracking (SPT) and fluorescence cross-correlation spectroscopy (FCCS) to investigate how membrane organization impacts the basal activity of the M ₁ muscarinic receptor (M ₁ R) and the adenosine A _2A receptor (A _2A R). M ₁ R showed minimal basal Ca ²⁺ responses, whereas A _2A R exhibited basal cAMP production that was eliminated by an inverse agonist. FCCS showed that in the absence of ligands only a small portion of M ₁ R co-diffused with its cognate Gα ₁₁ protein without ligand, whereas a larger fraction of A _2A R co-diffused with Gα _s . Interestingly, SPT revealed that A _2A R was enriched in slowly diffusing, confined states with spatial scales around 150–200 nm. The sensitivity to cholesterol- and membrane-order-altering agents suggests they are associated with raft-related membrane organization. Dual-color tracking and diffusion mapping demonstrated that A _2A R and Gα _s shared confinement domains under basal conditions, whereas M ₁ R and Gα ₁₁ only showed such co-confinement in the active state. We propose a model in which the co-confinement of GPCRs and G proteins within plasma membrane nanodomains promotes constitutive activity by increasing opportunities for transient productive encounters. This model links receptor conformational dynamics and nanoscale membrane organization to basal signaling output. One-sentence summary: Single-particle tracking experiments show that co-confinement of G protein-coupled receptors and G proteins within membrane nanodomains is an important factor for basal GPCR signaling.

Large proteins and dilute spin systems within a deuterated background are often characterized by long proton (¹H) spin-lattice relaxation times (T₁), which directly impacts the recycle delay and hence, the total experimental time. Dioxygen (O₂) is a well-known paramagnetic species whose short electronic spin-lattice relaxation time (7.5 ps) contributes to effective spin-lattice relaxation of high gamma nuclei. Oxygen’s chemical potential and high diffusivity also allows it to access both the protein exterior and much of the (hydrophobic) interior of the protein. Consequently, at O₂ partial pressures of ~ 10 bar, ¹H and ¹⁹F spin-lattice relaxation rates (R₁) typically reach 3–5 Hz (versus rates of 0.7-1.0 Hz without oxygen) with comparable line-broadening in protein NMR spectra. Using fluoroacetate dehalogenase (FAcD) a soluble 35 kDa homodimeric enzyme, a nanodisc-stabilized G protein-coupled receptor (A_2AR), and bovine serum albumin (BSA) as test cases, a 3-fold savings in time was achieved in acquiring ¹H-¹⁵ N HSQC and ¹⁹F NMR spectra, after oxygenation at 9 bar for 24 h. Additional spin-diffusion effects are anticipated to contribute to uniform ¹H spin-lattice relaxation for both solvent-exposed and buried protons, as demonstrated by T₁ relaxation analysis of amides in ¹⁵N-labeled FAcD. Finally, we show that in protein samples dissolved oxygen pre-equilibrated at 9 bar (pO₂) is largely retained in solution at 20° C or lower, using a standard NMR tube for a period of 3–4 days, thus avoiding the use of specialized apparatus or high-pressure NMR tubes in the spectrometer. The convenience of being able to add or remove the quenching species, while avoiding any complex apparatus in the NMR experiment, makes this a practical tool for both ¹⁹F, ¹H-¹³ C, and ¹H-¹⁵ N NMR studies of proteins.

5-Fluoroanthranilic acid-(phenyl-¹³C₆) is derived from ¹³C-enriched 4-fluoronitrobenzene and converted to the tryptophan analogue, allowing (¹⁹F,¹³C) TROSY spectroscopy (created with BioRender.com).

The adenosine A_2A receptor (A_2AR) engages several G proteins, notably G_o and its cognate G_s protein. This coupling promiscuity is facilitated by a dynamic ensemble, revealed by ¹⁹F nuclear magnetic resonance imaging of A_2AR and G protein. Two transmembrane helix 6 (TM6) activation states, formerly associated with partial and full agonism, accommodate the differing volumes of G_s and G_o. While nucleotide depletion biases TM7 toward a fully active state in A_2AR–G_s, A_2AR–G_o is characterized by a dynamic inactive/intermediate fraction. Molecular dynamics simulations reveal that the NPxxY motif, a highly conserved switch, establishes a unique configuration in the A_2AR–G_o complex, failing to stabilize the helix-8 interface with G_s, and adoption of the active state. The resulting TM7 dynamics hamper G protein coupling, suggesting kinetic gating may be responsible for reduced efficacy in the noncognate G protein complex. Thus, dual TM6 activation states enable greater diversity of coupling partners while TM7 dynamics dictate coupling efficacy.

The conformational features of alpha-halopropiophenones were investigated to understand the influence of alpha-halogens on conformation through hyperconjugative interactions, electrostatics, and steric factors. Using NMR, C-H scalar coupling constants were measured in different solvents, revealing a pattern in the conformational equilibria, which we validated by computational means. This behavior arises largely from hyperconjugative effects with the exception of the fluoro-derivatives, which are also influenced by steric and electrostatic interactions. In all cases, the contribution to hyperconjugation of the alpha-halo ketones is driven by the oxygen lone pair (rather than the C-X bond), which donates electron density to the adjacent C-C bonds. Additionally, C-C alpha bond rotation generates distortions in the side chain, responsible for destabilization, thus affecting system conjugation. These structural features identified for the alpha-halo ketones are also reflected in their reactivity, which is distinct from that expected for nucleophilic addition.