Long-range allosteric communication, double mutant cycles, and energetic coupling in SARS-CoV-2 spike protein

bioRxiv (Cold Spring Harbor Laboratory) · 2026-04-22

SARS-CoV-2 spike protein is continuously evolving, leading to new variants. While mutations in the receptor-binding domain (RBD) enhance binding to the ACE2 receptor and evade neutralizing antibodies, the function of mutations in the N-terminal domain (NTD) remains poorly understood. Using two independent methods, surface plasmon resonance (SPR) and cryo-EM, we show that NTD mutations increase the population of spike protein with the RBD in the "up" conformational state. SPR association and dissociation kinetics of spike binding to ACE2 and antibodies, analyzed using a coupled equilibrium model, determined the relative populations and indicated that the RBD-up-to-down transition is rate-limiting relative to the RBD-down-to-up transition. Advanced model fitting of cryo-EM Coulomb potential maps confirmed the populations. The combined effect of NTD and RBD mutations exceeds the sum of their individual effects, indicating long-range allosteric communication and energetic coupling within the spike protein.

BPS2026 – Long-range allosteric communication in SARS-CoV-2 spike protein

Biophysical Journal · 2026-02-01

Deacetylation of SOD3 by Sirtuins Restores Furin Cleavage

Free Radical Biology and Medicine · 2025-10-30

Synergistic effect of plunger insertion and siliconization methods on particle formation of an antibody formulation

Journal of Pharmaceutical Sciences · 2025-10-17

Magnetic Nanoworm-Based Screening of Yeast Library for Anti-PEG Nanobodies

Molecular Pharmaceutics · 2025-07-02

PEGylation is commonly utilized to modify nanoparticles, vaccines, therapeutic proteins, and biomaterials. The increased use of PEG-containing cosmetics and medicines results in anti-PEG immunoglobulins in humans, including IgG, IgM, and IgE types. Recent studies have shown that high-affinity antibodies against PEG can be isolated through immunization. These antibodies have been used in immunoassays and for decorating PEGylated nanoparticles with targeting ligands. However, conventional antibodies have limitations, such as their large size and potential stability issues. Here, we developed a protocol to isolate anti-PEG nanobodies (Nbs) from a yeast library. We prepared ∼60 nm fluorescently labeled PEGylated and non-PEGylated iron oxide nanoworms for negative and positive magnetic selection, followed by multiparameter flow cytometry sorting. The representative Nb clone exhibited highly specific binding to PEGylated liposomes and nanoparticles and was able to compete with commercial antibackbone PEG IgG but not with anti-methoxy PEG IgG for PEG binding. The Nb showed a micromolar dissociation constant, no aggregation, high thermal stability, and reversible structural stability. This work represents a proof-of-concept screening and isolation of anti-PEG Nbs.

Polysorbates and poloxamer rescue filter-induced serotype-dependent loss of AAVs

Journal of Pharmaceutical Sciences · 2025-05-09 · 1 citations

Effect of silicone oil on the structure, stability, and aggregation of a therapeutic antibody

Journal of Pharmaceutical Sciences · 2025-05-23 · 9 citations

Functional differences in the mu opioid receptor <scp>SNP 118A</scp>&gt;G are dependent on receptor splice‐variant and agonist‐specific recruitment of β‐arrestin

Clinical and Translational Science · 2024-08-01 · 2 citations

Abstract The OPRM1 gene codes for the mu opioid receptor (MOR) and polymorphisms are associated with complex patient clinical responses. The most studied single nucleotide polymorphism (SNP) in OPRM1 is adenine (A) substituted by guanine (G) at position 118 (118A&gt;G, rs1799971) leading to a substitution of asparagine (Asn) for aspartic acid (Asp) at position 40 in the N terminus of the resulting protein. To date, no structural explanation for the associated clinical responses resulting from the 118A&gt;G polymorphism has been proposed. We utilized computational modeling paired with functional cellular assays to predict unstructured N‐ and C‐terminal regions of MOR‐1. Using molecular docking and post‐docking energy minimizations with morphine, we show that the extracellular substitution of Asn at position 40 alters the cytoplasmic C‐terminal conformation, while leaving the G‐protein binding interface unaffected. A real‐time BRET assay measuring G‐protein and β‐arrestin association with MOR r generated data that tested this prediction. Consistent with this in silico prediction, we show changes in morphine‐mediated β‐arrestin association with receptor variants with little change in morphine‐mediated G‐protein association comparing MOR‐1 wild type (WT) to MOR‐1 118A&gt;G . We tested the system with different opioid agonists, the OPRM1 118A&gt;G SNP, and different MOR splice variants (MOR‐1 and MOR‐1O). These results are consistent with the observation that patients with the 118A&gt;G OPRM1 allele respond more readily to fentanyl than to morphine. In conclusion, the 118A&gt;G substitution alters receptor responses to opioids through variable C‐terminal domain movements that are agonist and splice variant dependent.

Biophysical characterization of the dystrophin C-terminal domain: Dystrophin interacts differentially with dystrobrevin isoforms

Journal of Biological Chemistry · 2024-11-17 · 1 citations

Duchenne muscular dystrophy (DMD) gene encodes dystrophin, a large multidomain protein. Its nonfunctionality leads to dystrophinopathies like DMD and Becker muscular dystrophy, for which no cure is yet available. A few therapies targeted towards specific mutations can extend the lifespan of patients, although with limited efficacy and high costs, emphasizing the need for more general treatments. Dystrophin's complex structure with poorly understood domains and the presence of multiple isoforms with varied expression patterns in different tissues pose challenges in therapeutic development. The C-terminal (CT) domain of dystrophin is less understood in terms of its structure-function, although it has been shown to perform important functional roles by interacting with another cytosolic protein, dystrobrevin. Dystrophin and dystrobrevin stabilize the sarcolemma membrane by forming a multiprotein complex called dystrophin-associated glycoprotein complex that is destabilized in DMD. Dystrobrevin has two major isoforms, alpha and beta, with tissue-specific expression patterns. Here, we characterize the CT domain of dystrophin and its interactions with the two dystrobrevin isoforms. We show that the CT domain is nonglobular and shows reversible urea denaturation as well as thermal denaturation. It interacts with dystrobrevin isoforms differentially, with differences in binding affinity and the mode of interaction. We further show that the amino acid differences in the CT region of dystrobrevin isoforms contribute to these differences. These results have implications for the stability of dystrophin-associated glycoprotein complex in different tissues and explain the differing symptoms associated with DMD patients affecting organs beyond the skeletal muscles.

Isothermal titration calorimetry and surface plasmon resonance methods to probe protein–protein interactions

Methods · 2024-03-15 · 30 citations