The target of the new work is the α subunit of the Gi protein, a two-lobed molecular machine. One lobe, the Ras-like domain, carries the guanine nucleotide pocket that holds GDP in the resting state. The other, the α-helical domain, is a six-helix bundle that clamps down over that pocket, burying GDP at the interface between the two lobes. Activation by a receptor requires the helical domain to swing open by tens of angstroms, exposing the nucleotide site so GDP can escape and GTP can flood in. That opening step, long inferred from crystallography and double electron–electron resonance spectroscopy, had never been watched in real time on single molecules, and its dependence on the drug occupying the receptor was unknown.

To capture those motions, the team engineered a minimal-cysteine version of the Gαi1 α subunit and attached two fluorophores, LD555 and JFX673, at positions R90C on the helical domain and E238C on the Ras-like domain. When the helical domain opens, the dyes drift apart and the Förster resonance energy transfer signal between them drops; when the domain closes, the signal rises. The labeled heterotrimers were coupled to purified μ-opioid receptors, immobilized on quartz slides through an anti-Flag antibody fragment, and imaged with total internal reflection fluorescence microscopy at 50-millisecond resolution. The construct behaved like the wild-type protein in GTP turnover assays, preserving the canonical rank order of ligand efficacies, so the dynamics observed could be interpreted with confidence.

Individual complexes flickered reversibly between a low-FRET, open state centered near 0.27 and a high-FRET, closed state near 0.62, exchanging roughly once per second. The crucial finding emerged when the researchers compared complexes bound to different opioids. Full agonists such as lofentanil, BU72 and DAMGO shifted the equilibrium toward the open state, while partial agonists such as morphine, buprenorphine and mitragynine pseudoindoxyl left the protein spending more time closed. Strikingly, the fraction of time spent in the open state correlated almost perfectly with GTP turnover activity, the standard biochemical readout of efficacy, with a coefficient of determination of 0.97. The unliganded receptor was least able to stabilize the open conformation, matching its sluggish basal activity.

Hidden Markov modeling of the trajectories revealed where the ligand exerts its leverage. Dwell times in the open state were essentially identical across all ligands, averaging about 1.4 seconds, but dwell times in the closed state depended strongly on efficacy. Lofentanil-bound complexes opened roughly 2.25-fold faster than unliganded ones, at about 0.9 per second versus 0.4 per second. In other words, high-efficacy drugs do not hold the G protein open; they destabilize its closed, GDP-clamped configuration, making domain separation more likely. A transient mid-FRET state near 0.47, often lasting barely 100 milliseconds, appeared to serve as an unstable intermediate on the path between the extremes.

The generality of the mechanism was tested with a second receptor. Neurotensin receptor 1, coupled to the same labeled Gi protein, produced an analogous correlation between open-state occupancy and GTP turnover, with R² of 0.89. Adding SBI-553, an allosteric modulator known to bias neurotensin signaling, shifted the helical domain further toward the open state in proportion to its positive effect on nucleotide exchange. Together with earlier bioluminescence resonance energy transfer studies showing ligand-specific G protein conformations at calcitonin and α2-adrenergic receptors, the results suggest that long-range allosteric encoding of efficacy may be a widespread feature of GPCR signaling rather than an opioid peculiarity.

The team also dissected the nucleotide chemistry underlying these motions. Because GTP or GDP delivery causes the complex to fall apart too quickly for equilibrium imaging, the researchers used a mixture of GMP and pyrophosphate, which together mimic the triphosphate in the nucleotide pocket without triggering dissociation. Titrating this pair onto DAMGO-bound complexes drove a concentration-dependent closure of the helical domain, confirming that the closed state binds nucleotide more tightly than the open state. Morphine-bound complexes were roughly fourfold more sensitive to the nucleotide mimic than DAMGO-bound ones, with half-maximal effective concentrations of 0.34 and 1.53 micromolar respectively, exactly as expected if partial agonists leave the G protein in a higher-affinity, more closed ensemble. Synchronization analysis caught many closure events passing through the partially open intermediate, implying that nucleotide contacts are broken and formed progressively rather than all at once, an energetically economical route for exchange.

These dynamics resolve a long-standing pharmacological puzzle. In isolation, buprenorphine-bound complexes actually bound the fluorescent GTP analog GTPγS faster than lofentanil-bound complexes, which seems backwards for a weaker agonist. But the cytoplasm contains both GDP and GTP, with GTP about tenfold more abundant, so activation is limited by GDP release, not GTP binding. When physiological GDP was added, the ranking reversed: GDP competitively slowed GTP loading onto buprenorphine-bound complexes far more than onto lofentanil-bound ones. Full agonists pry the domains apart, lower nucleotide affinity and let GDP escape quickly; partial agonists favor closure, prolong GDP residence and delay activation. Structurally, the authors trace this allostery to the α5 helix of Gα, which inserts into the receptor’s cytosolic vestibule and disrupts the α1 helix and the interdomain interface, with full agonists promoting deeper α5 engagement through their larger displacement of transmembrane helix 6.

The implications reach well beyond basic mechanism. Full agonists dominate pain management, while partial agonists such as buprenorphine anchor the treatment of opioid use disorder, and a mechanistic account of why partial agonism arises, kinetic trapping of GDP-bound intermediates rather than mere failure of receptor coupling, gives medicinal chemists a new dial to tune. If the G protein itself is a locus where ligand-specific signaling behavior is encoded, then safer analgesics might be designed not only by shaping receptor conformations but by controlling how those conformations propagate into the transducer. The study also cautions structural biologists: cryo-electron microscopy snapshots, which capture the lowest-energy states of a heterogeneous ensemble, often show minimal differences between ligands, whereas the single-molecule trajectories reveal that subtle changes in interdomain energetics can transform the signaling outcome. Watching the machine move, it turns out, matters as much as photographing where it rests.

Subject of Research: Allosteric control of G protein conformational dynamics by opioid ligands at the μ-opioid receptor

Article Title: Ligand-dependent G protein dynamics underlying opioid efficacy

Article References: Ligand-dependent G protein dynamics underlying opioid efficacy. (n.d.). https://doi.org/10.1038/s41594-026-01893-6

Image Credits: AI Generated

DOI: 10.1038/s41594-026-01893-6

Keywords: μ-opioid receptor, G protein, GPCR, single-molecule FRET, allostery, opioid efficacy, GDP release, nucleotide exchange, buprenorphine, pharmacology, conformational dynamics, structural biology

The Society cited two pillars of Puglisi’s scientific career as the basis for the honor. The first is a body of sophisticated nuclear magnetic resonance, or NMR, studies that have advanced fundamental concepts of how RNA is recognized by other molecules. The second is a series of groundbreaking single-molecule experiments using fluorescence resonance energy transfer, commonly known as FRET, that have illuminated the inner workings of the ribosome, the molecular machine that translates genetic information into proteins. Together, these contributions have shaped how an entire generation of researchers thinks about RNA structure, dynamics, and function.

The award’s namesake carries deep personal significance in this case. Ignacio “Nacho” Tinoco, after whom the prize is named, was Puglisi’s mentor, and his contributions to the spectroscopic, thermodynamic, structural, and single-molecule study of RNA are considered foundational to the modern understanding of the physical principles governing macromolecules. Tinoco challenged the biophysics community to continually push the boundaries of fundamental understanding, and the award created in his memory recognizes scientists who carry that spirit forward. That Puglisi, a direct scientific descendant of Tinoco, should now receive the honor bearing his mentor’s name gives the 2027 announcement a rare narrative symmetry.

Biophysical Society President Karen Fleming of Johns Hopkins University praised Puglisi’s originality and the depth of his influence. “Jody is one of the most creative and innovative scientists in the field of translation,” Fleming said. “His recognition aptly honors the legacy of Ignacio “Nacho” Tinoco, Jody’s mentor, who challenged our community to continually push our fundamental understanding of biophysics.” Fleming added that Puglisi’s contributions are unique, noting that his research over the past decades has provided physical and structural foundations for RNA’s dynamic role in biology and has revealed many of the most complex yet fundamental properties of RNA-mediated biology.

To appreciate why Puglisi’s NMR work earned such recognition, it helps to understand what the technique makes possible. NMR spectroscopy allows researchers to probe the structure and motion of molecules at atomic resolution in solution, closer to the conditions of a living cell than many other structural methods. RNA, however, is notoriously difficult to study this way. Unlike proteins, many RNA molecules are flexible, dynamic, and conformationally heterogeneous, adopting ensembles of shapes rather than a single rigid structure. Extracting meaningful structural and dynamic information from such systems demands both methodological ingenuity and deep physical insight, qualities that have characterized Puglisi’s approach throughout his career.

Through decades of NMR studies, Puglisi and his collaborators helped establish how RNA molecules are recognized by binding partners, including proteins and small molecules. RNA recognition underlies a vast range of biological processes, from the regulation of gene expression to the action of antibiotics that target bacterial ribosomes. By defining the structural principles that govern these interactions, his work provided a conceptual framework that other laboratories have built upon, connecting atomic-level detail to the broader mechanics of molecular biology. This kind of fundamental knowledge is also a starting point for practical applications, since many drugs work by binding to RNA or to the molecular machines that interact with it.

The second strand of Puglisi’s recognized work, single-molecule FRET, represents a different but complementary way of seeing biology. In a FRET experiment, two fluorescent tags are placed on a molecule or molecular complex at carefully chosen positions. When the tags come close together, energy transfers between them, producing a signal that reports on the distance between the labeled sites. By monitoring individual molecules one at a time, researchers can watch molecular machines move in real time, capturing transient states and rare events that are invisible to methods that average signals over enormous populations of molecules. For something as dynamic as the ribosome, this capability is transformative.

Using these single-molecule approaches, Puglisi’s laboratory has dissected some of the most intricate behaviors of the ribosome during translation, the process by which messenger RNA is decoded into amino acid chains. The Society’s announcement specifically highlighted his studies of translational frameshifting, bypassing, stalling, and pausing during elongation. Frameshifting occurs when the ribosome slips by one or more nucleotides, reading the genetic message in a different frame and producing a different protein, a mechanism that certain viruses exploit and that cells use for regulation. Bypassing, meanwhile, allows a ribosome to skip over stretches of mRNA, while stalling and pausing during elongation influence how quickly and accurately proteins are made. Observing these events directly, one ribosome at a time, revealed kinetic details and intermediate states that population-level experiments simply could not resolve.

What makes this body of work especially significant is how it reframed the ribosome from a static structure into a dynamic machine. High-resolution structures had provided stunning snapshots of the ribosome at work, but the transitions between those states, their timing, and their probabilities remained largely hidden. Single-molecule FRET brought those dynamics into view, showing how the ribosome samples different conformations, how transfer RNAs move through the machine, and how the messenger RNA itself can be handled in unexpected ways. In doing so, Puglisi’s research connected the physical chemistry of molecular motion to one of biology’s most essential processes, the synthesis of every protein in every living cell.

The Biophysical Society, founded in 1958, is a professional scientific society established to lead a global community working at the interface of the physical and life sciences, across all levels of complexity, and to foster the dissemination of that knowledge. With roughly 6,000 members located around the world, the Society promotes growth in the field through its Annual Meeting, its publications, and its outreach activities, and its members teach and conduct research in colleges, universities, laboratories, government agencies, and industry. The Tinoco Award, presented within that community, honors a legacy of rigorous, quantitative inquiry into the molecules of life. When Puglisi accepts the 2027 prize in Philadelphia, the moment will recognize not only an individual career of technical brilliance and conceptual clarity, but also a scientific lineage running from Tinoco’s pioneering studies of RNA physical chemistry to the single-molecule era in which the movements of individual ribosomes can be watched as they read the genetic code.

Subject of Research: Physical chemistry of RNA and ribosome function recognized by the Biophysical Society's 2027 Ignacio Tinoco Award

Article Title: Joseph D. (Jody) Puglisi to receive the 2027 Ignacio Tinoco Award in Physical Chemistry of Macromolecules

Article References: Joseph D. (Jody) Puglisi to receive the 2027 Ignacio Tinoco Award in Physical Chemistry of Macromolecules. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: Joseph Puglisi, Biophysical Society, Ignacio Tinoco Award, NMR spectroscopy, single-molecule FRET, RNA recognition, ribosome, translation, translational frameshifting, physical chemistry, macromolecules, Stanford University