Biology subjects

Lee, A. L.

Publications and source records attributed to Lee, A. L..

7 recordsLinked to original sources

bioRxiv2026

Dynamic view of an allosteric intermediate in a positively cooperative dimer

Positive cooperativity in ligand binding is a hallmark of allosteric oligomers, yet how the first binding event enhances the second remains obscure, because the pivotal singly-bound intermediate (lig) is thermodynamically disfavored and rarely accumulates. Distinguishing concerted (MWC) from sequential (KNF) mechanisms turns on one question: when a ligand binds one protomer, does its empty partner change conformation? Here we resolve this for the allosteric homodimer chorismate mutase (CM) using mixed-labeled dimers (MLDs), in which a single maleimide crosslink stabilizes a heterodimer carrying one NMR-labeled and one active-site-inactivated subunit, trapping lig for prolonged study. Isothermal titration calorimetry shows that inhibitor binding to CM is positively cooperative and entirely entropy-driven, with the second event carrying far larger enthalpic and entropic swings than the first. Protomer-resolved NMR reveals that the first binding event switches both subunits to the relaxed (R) state--a concerted, MWC-like transition that rules out a strictly sequential model--yet the empty subunit is not a clean R conformer but a "fuzzy", dynamically heterogeneous ensemble, with extensive microsecond- millisecond motion focused at the dimer interface, and a raft of residues surrounding the empty active site. Backbone probes confirm that both critical 11-12 loops adopt their active posture upon first ligand binding. The mismatch between the chemical-shift picture (MWC-like) and the thermodynamics (weighted toward the second event) argues that cooperativity is not encoded by a simple two-state switch, but by activated dynamics that a purely structural model cannot capture. Significance StatementCooperative ligand binding underlies allosteric control across biology, but mechanisms have been hard to pin down because the crucial half-bound intermediate of a cooperative dimer barely exists at equilibrium. We stabilize this intermediate in the enzyme chorismate mutase by chemically linking one NMR-visible subunit to one that cannot bind ligand, letting us watch each subunit independently. Binding the first ligand flips both subunits to the active state, yet the empty subunit becomes highly dynamic rather than rigidly active. Combined with calorimetry, this shows that the free energy of cooperativity must be stored in ensemble dynamics, not merely in a switch between two structures. The linkage method further opens the door to additional solution-based studies required to better understand allostery and benchmark tools of the future.

biophysics↗

bioRxiv2026

Incidental conformational switching in an allosteric enzyme

The classical understanding of allostery was initially grounded in two-state models, such as MWC and KNF, where structure and function are inextricably linked through transitions between low-(T) and high-affinity (R) states. Here, we show Yeast chorismate mutase (CM) provides a vivid example of the growing list of exceptions to the traditional T vs R two-state allosteric paradigm. While CM exhibits dynamic sampling of the R-state in the presence of the activator tryptophan (Trp), suggesting a conformational selection (CS) mechanism, we present multiple instances where conformational status and catalytic activity are decoupled. Using NMR spectroscopy and kinetic assays, we identify CM variants that reside almost exclusively in the T conformation can exhibit maximal activity, while others that predominantly occupy the R conformation are weakly active. Quantitative comparison of experimental data with a parameterized CS model reveals deviations of up to two orders of magnitude, ruling out the simplest two-state model for substrate affinity modulation in this system. We propose that the observed T-to-R switching in CM is "incidental", a byproduct of an evolved energy landscape that allows access to the substrate-bound pose but does not mechanistically determine affinity. Our findings suggest that allosteric regulation in CM may instead be driven by local features of the ground-state ensemble, which operate independently of global T/R status. This work further highlights an emerging view that the mere observation of a pre-sampled active conformation does not sufficiently prove a two-state mechanism and further underscores the need for deeper ensemble-based perspectives in protein engineering and allostery.

biophysics↗

bioRxiv2026

HaloMPNN: retraining ProteinMPNN on halophilic proteomes for salt-tolerant enzyme design

Machine learning-guided protein sequence redesign is now routinely used to optimize multiple properties relevant for protein engineering, most prominently thermostability and recombinant expression levels. Salt tolerance is a valuable property for "blue-biotechnology"- enabled biomanufacturing, yet no generative computational method exists to redesign proteins for increased salt tolerance. We hypothesized that a training dataset heavily biased toward salt-adapted proteomes would yield a model capable of designing proteins with halophilic properties. To test this, we retrained the sequence redesign model ProteinMPNN on proteins from "salt-in" extreme halophiles such as Haloarcula marismortui, a Dead Sea archaeon that grows optimally near 3-4 M NaCl, roughly six times the salinity of seawater, and accumulates molar concentrations of salts in its cytoplasm. Our model, HaloMPNN, redesigns non-halophilic proteins so that their properties shift towards those of natural halophilic proteins: lower predicted isoelectric point, greater surface acidity, and reduced surface and core hydrophobicity. Redesigning a broad range of non-halophilic proteins with SolubleMPNN, ProteinMPNN, and HyperMPNN shows that this shift is specific to HaloMPNN rather than a generic consequence of sequence redesign. HaloMPNN therefore offers both a route to designing candidate salt-tolerant enzymes and a means of identifying the characteristics that underlie halophilic adaptation.

bioengineering↗

bioRxiv2026

Zero-shot ecological annotation of microbial genomes with myLLannotator accelerates scientific discovery

Large language models (LLMs) are promising scientific assistants, but many models remain inaccessible for resource-constrained scientists. To address this, we present myLLannotator, a python package for metadata annotation built on llama3.2-3B. myLLannotator labels 18,000+ genomes in ~2 hours on a laptop, reproducing a discovery involving substantial manual annotation. We use myLLannotator to discover that duplicated genes are depleted in endosymbiotic bacteria, demonstrating its power to accelerate hypothesis testing and discovery.

bioinformatics↗

bioRxiv2022

Allosteric inactivation of an engineered optogenetic GTPase

Optogenetics is a technique for establishing direct spatiotemporal control over molecular function within living cells using light. Light application induces conformational changes within targeted proteins that produce changes in function. One of the applications of optogenetic tools is an allosteric control of proteins via light-sensitive LOV2 domain, which allows direct and robust control of protein function. Computational studies supported by cellular imaging demonstrated that application of light allosterically controlled signaling proteins Vav2, ITSN, and Rac1, but the structural and dynamic basis of such control has yet to be elucidated by experiment. Here, using NMR spectroscopy, we discover principles of action of allosteric control of cell division control protein 42 (CDC42), a small GTPase involved in cell signaling. Both LOV2 and Cdc42 employ flexibility in their function to switch between "dark"/ "lit" or active/inactive states, respectively. By conjoining Cdc42 and LOV2 domains into the bi-switchable fusion Cdc42Lov, application of light - or alternatively, mutation in LOV2 to mimic light absorption - allosterically inhibits Cdc42 downstream signaling. The flow and patterning of allosteric transduction in this flexible system is well-suited to observation by NMR. Close monitoring of the structural and dynamic properties of dark versus lit states of Cdc42Lov revealed lit-induced allosteric perturbations. Chemical shift perturbations for lit mimic, I539E, have distinct regions of sensitivity and both the domains are coupled together leading to bi-directional interdomain signaling. Insights gained from this optoallosteric design will increase our ability to control response sensitivity in future designs. Significance StatementControl of cell signaling activity in proteins by light is one of the primary goals of optogenetics. The hybrid light-receptor/cell-signaling protein Cdc42Lov was engineered recently as an optogenetic tool, employing a novel allosteric strategy that results in photoinhibition. In contrast to previous activation designs, the mechanism of inhibition of GTPase signaling activity in Cdc42 is only apparent at a detailed structural and dynamic level. NMR characterization of dark and mutationally "lit" forms reveals the allosteric interdomain perturbations, knowledge of which will enhance future applications of this design strategy.

biophysics↗

bioRxiv2021

CuAAC stabilization of an NMR mixed labeled dimer

Homo dimers are the most abundant type of enzyme in cells and as such, they represent the archetypal system for studying the remarkable phenomenon of allostery. In these systems, in which the allosteric features are manifest by the effect of the first binding event on the similar event at the second site, the most informative state is the asymmetric single bound (lig1) form, yet it tends to be elusive thermodynamically. Here we take significant steps towards obtaining milligram quantities of pure lig1 of the allosteric homodimer, chorismate mutase, in the form of a mixed isotopically labeled dimer stabilized by Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) between the subunits. Below, we outline several critical steps required to generate high yields of both types of unnatural amino acid-containing proteins, and overcome multiple pitfalls intrinsic to CuAAC to obtain high yields of pure, fully intact, and active mixed labeled dimer. These data not only will make possible NMR-based investigations of allostery envisioned by us, but should also facilitate other structural applications where specific linkage of proteins is helpful.

biophysics↗

bioRxiv2020

Hyaluronic Acid on the Urokinase Sustained Release with a Hydrogel System Composed of Poloxamer 407: Enhanced Stability of Intracellular Interaction by Adding HA.

Pleural empyema is an inflammatory condition characterized by accumulation of pus inside the pleural cavity, which is usually followed by bacterial pneumonia. During the disease process, the pro-inflammatory and pro-fibrotic cytokines in the purulent pleural effusion cause proliferation of fibroblasts and deposition of extracellular matrix, which lead to fibrin deposition and fibrothorax. Urokinase instillation therapy through a chest drainage tube is frequently used for fibrinolysis in patients with empyema. However, urokinase treatment requires multiple instillation (2-3 times per day, for 4-8 days) and easily flows out from the chest drainage tube due to its high water solubility. In this in vitro study, we developed a thermo-responsive hydrogel based on poloxamer 407 (P407) combined with hyaluronic acid (HA) for optimal loading and release of urokinase. Our results show that the addition of HA to poloxamer gels provides a significantly more compact microstructure, with smaller pore sizes (**p < 0.001). The differential scanning calorimetry (DSC) profile revealed no influence on the micellization intensity of poloxamer gel by HA. The 25% poloxamer-based gel was significantly superior to the 23% poloxamer-based gel, with slower gel erosion when comparing the 16th hour residual gel weight of both gels (*p < 0.05; **p < 0.001). The 25% poloxamer-HA gel also exhibited a superior urokinase release profile and longer release time. A Fourier-transform infrared spectroscopy (FT-IR) study of the P407/HA hydrogel showed no chemical interactions between P407 and HA in the hydrogel system. The thermoresponsive P407/HA hydrogel may have a promising potential in the loading and delivery of hydrophilic drugs. On top of that, in vitro toxicity test of this combination demonstrates a lower toxicity.

pharmacology and toxicology↗