Wiley: Protein Science: Table of Contents

SNAC‐DB: An ML‐ready database for antibody and NANOBODY® VHH–antigen complexes with expanded structural diversity and real‐world benchmarking

Sun, 07 Jun 2026 22:46:00 -0700

Abstract

Predicting antibody and NANOBODY® VHH–antigen complexes remains a critical challenge for state-of-the-art structure prediction models, limiting their impact in therapeutic discovery pipelines. We introduce SNAC-DB, an ML-ready database and curation pipeline enriched with structural biology expertise, designed to accelerate model accuracy and generalization by providing 31–37% expanded structural diversity over existing resources like SAbDab through comprehensive re-curation that extracts maximum value from available experimental structures. SNAC-DB expands coverage by capturing often-overlooked complexes and accurately identifying complete multi-chain epitopes through improved biological-assembly based logic. Built for ML practitioners, SNAC-DB provides standardized formats with multi-threshold structure-based clustering to enable principled sample weighting during training. Using a rigorous benchmark of public PDB entries deposited post-May 2024 plus confidential therapeutic structures, we evaluate seven leading models (Protenix-v1, OpenFold-3p2, RosettaFold-3, Boltz-2, Boltz-1x, Chai-1, and AlphaFold2.3-multimer) with evaluation methodology tailored to antibody/NANOBODY® VHH–antigen complexes to ensure correct handling of multi-chain epitopes, revealing systematic performance gaps: success rates rarely exceed 25%, confidence-based ranking fails to identify best predictions even when accurate structures exist in ensembles, and all models consistently struggle with therapeutically relevant NANOBODY® VHHs. Systematic evaluation of sampling strategies demonstrates that while generating 1000 samples per target substantially increases the likelihood of producing accurate structures (oracle selection improves from 11.9% to 50.5%), confidence-based ranking remains nearly flat (between 10.9% and 14.9%), revealing that improved ranking mechanisms represent a more tractable path to performance gains. Finally, fine-tuning GeoDock on SNAC-DB yields higher success rates than training on SAbDab (11.0% vs. 7.1% for antibodies; 7.0% vs. 4.0% for NANOBODY® VHHs), suggesting that SNAC-DB's expanded structural diversity translates to improved model generalization.

PolyProline Predictor: A web server for empirical sequence‐based prediction of polyproline II helices

Rubén López‐Sánchez, David Pantoja‐Uceda, Miguel Mompeán, Douglas V. Laurents — Sun, 07 Jun 2026 20:54:54 -0700

Abstract

Polyproline II (PPII) helices are extended left-handed secondary structures increasingly recognized for their roles in molecular recognition, signaling and within intrinsically disordered regions of proteins. Despite their functional importance, predicting regions with propensity to form PPII helices from sequence alone remains challenging due to subtle sequence determinants and their frequent misclassification as random coil. Here, we present PolyProline Predictor (PPP), a user-friendly web server (https://rmni.iqf.csic.es/software/polypropre/) for empirical, sequence-based prediction of PPII helices. Unlike machine learning approaches, PPP aligns query sequences against a curated database of experimentally validated PPII helices, providing an interpretable, composition-, and position-sensitive similarity map. PPP successfully identified conserved PPII motifs in diverse proteins, and predicted the presence of similar motifs in regions lacking experimental structures but modeled by AlphaFold as extended PPII conformations, such as glycine-rich plant proteins, mycobacterial PE_PGRS virulence factors, and the “disordered” C-terminal tails of GroEL and its homologs, as well as the amyloid-flanking region of the necroptosis effector RIPK3. Molecular dynamics simulations further supported persistent PPII helical bundles in three glycine-rich mycobacterial proteins and more heterogeneous, transient PPII populations in plant proteins and RIPK3. Circular dichroism and nuclear magnetic resonance (NMR) spectroscopy validated these predictions for RIPK3, revealing partially populated PPII conformations flanking its amyloid core. Such motifs may regulate its amyloid assembly, offering structural insight into mechanisms of functional amyloid formation. By combining experimental evidence with interpretable prediction, PPP fills a critical gap in bioinformatics tools and enables systematic exploration of regions with propensity to form PPII helices across proteomes, redefining the structural landscape of low-complexity regions.

Differential effects of lipid composition on the thermal and functional properties of membrane associated CYP2J2

Rajatabha Das, Henry M. Mastrion, Harrison B. Vassar, Aditi Das — Sat, 06 Jun 2026 05:49:41 -0700

Abstract

CYP2J2 is a membrane-bound cytochrome P450 that is expressed in cardiomyocytes, where it is known to metabolize arachidonic acid into cardioprotective epoxyeicosatrienoic acids (EETs). It consists of transmembrane domains embedded in the hydrophobic segments of the cell membrane and surrounded by various lipids. Currently, we lack a detailed understanding of the role of specific lipids in mediating the physicochemical properties of CYP2J2 and the factors that govern this phenomenon. In this study, CYP2J2 was reconstituted into nanodiscs with different lipid compositions, selected to reflect those of the endoplasmic reticulum (ER) membrane, where the enzyme is expressed. Using a combination of Nano-Differential Scanning Fluorimetry (nano-DSF) and UV–Visible Spectroscopy, we demonstrate that CYP2J2 undergoes a transition in its unfolding behavior between the detergent micelle and the nanodisc environment, with the first melting transition corresponding to heme perturbation. Furthermore, we show that altering the lipid environment causes shifts of up to 3–4°C and 8–9°C in the first and second melting transition temperatures, respectively, with sphingomyelin- and POPS (1-palmitoyl-2-oleoyl-glycero-3-phosphoserine) containing nanodiscs exhibiting the highest and lowest thermal stabilities, respectively. Lipid composition was found to have no effect on substrate (ebastine) binding affinities. However, NADPH-oxidation rates showed that lipid composition directly affects CYP2J2 function in nanodiscs by altering the rate of electron transfer between the CYP and its redox partner, Cytochrome P450 Reductase (CPR). Fluorescence anisotropy measurements with DPH (1,6-Diphenyl-1,3,5-hexatriene) were also used to characterize the membrane fluidity of cholesterol- and sphingomyelin-containing nanodiscs. Together, the results show that lipid composition directly modulates the thermal stability and functional properties of CYP2J2 in nanodiscs and underscore the importance of the charge of the lipid headgroup and membrane fluidity in our understanding of the mechanism by which lipid composition exerts these effects.

Machine‐learning prediction of affinity and epistasis in the bovine pancreatic trypsin inhibitor–chymotrypsin complex

Noam Tzuri, Itamar Kass, Yaron Orenstein, Niv Papo — Fri, 05 Jun 2026 04:50:26 -0700

Abstract

Protein–protein interactions (PPIs) are shaped by evolutionary pressures that fine-tune binding affinities and drive the epistatic relationships that support functional outcomes. Here, we used the complex of bovine pancreatic trypsin inhibitor (BPTI) and chymotrypsin as a model system to study how mutations at one or two positions affect binding affinity and epistasis. To predict the binding affinity landscape of the BPTI–chymotrypsin complex, we combined deep sequencing data, obtained from a saturation scanning mutagenesis BPTI library, with a machine-learning (ML) model. Using this ML model, which was trained on a subset of experimental binding data, we predicted the binding affinities and epistatic interactions across thousands of single and double BPTI mutants, including those not observed in the library. Our predictive approach completed missing data points and enabled us to reveal global trends in affinity changes and mutation couplings within specific binding interface positions. Our analysis revealed that different mutations in the same position may have different effects on affinity, with most double mutations leading to increased epistasis, particularly at hotspot positions, thereby indicating a cooperative binding effect. In most cases, affinity and epistasis were inversely correlated, with affinity enhancement of double-mutant variants being associated with negative epistasis. Our approach can be readily generalized to predict mutation effects in larger combinatorial libraries and in proteins for which structural information is lacking.

Correction to “How hydrophobicity, side chains, and salt affect the dimensions of disordered proteins”

Thu, 04 Jun 2026 21:55:36 -0700

Protein Science, Volume 35, Issue 7, July 2026.

Regulation of mitochondrial protein translocases

Lea Bertgen, Agnieszka Chacinska — Thu, 04 Jun 2026 04:22:26 -0700

Abstract

Mitochondria are essential for cellular health, and their function is underlain by the plasticity of the mitochondrial proteome. Most mitochondrial proteins are nuclear encoded, synthesized in the cytosol, and require precise import into mitochondrial subcompartments to fulfill their proper functions. Multimeric mitochondrial translocases ensure accurate protein localization and membrane integration. Recent work has begun to reveal how translocase activity and composition are dynamically regulated within mammalian cells. This review discusses regulatory mechanisms, including phosphorylation and protein degradation, that emerge as important players in adjusting the capacity and/or selectivity of the mitochondrial translocase to metabolic demands. Particular emphasis will be placed on the TIM23 complex as an emerging regulator of the inner membrane and matrix proteome composition.

Correction to “Aggregation‐driven expression of liraglutide precursors using engineered mini‐tags in Escherichia coli”

Wed, 03 Jun 2026 03:01:51 -0700

Protein Science, Volume 35, Issue 7, July 2026.

Correction to “Intrinsically dominant conformational diversity in PDZ1 within the tandem PDZ1–PDZ2 of human syntenin‐1 underlain by crystal structures”

Wed, 03 Jun 2026 02:52:55 -0700

Protein Science, Volume 35, Issue 7, July 2026.

Redox signals and oxidative stress in the control of mitochondrial protein import

Tue, 02 Jun 2026 21:38:14 -0700

Abstract

Mitochondrial protein import is essential for organelle biogenesis and cellular homeostasis. It operates in an environment that is intrinsically shaped by redox chemistry. Mitochondria are major sources of reactive oxygen species (ROS), which arise as by-products of oxidative phosphorylation. Cells therefore maintain sophisticated ROS-handling systems, including compartmentalized antioxidant networks, to balance redox signaling with protection from oxidative stress. Increasing evidence indicates that these redox conditions directly influence mitochondrial protein import at multiple levels. In this review, we provide an overview of ROS production, ROS signaling, and oxidative stress in relation to mitochondrial protein import. We outline the major mitochondrial protein import pathways, and discuss how their activity is modulated by redox-dependent mechanisms. A particular focus is placed on the mitochondrial disulfide relay system of the intermembrane space, which directly couples protein import to redox chemistry through oxidative folding, and how it is influenced by the local redox environment. Collectively, we propose that mitochondrial protein import is partially governed by redox-dependent mechanisms, enabling integration of metabolic state, stress responses, and signaling pathways.

Understanding chemical reactions through multimedia resolution data artistic educational tool (MRDAET)

Tue, 02 Jun 2026 21:35:40 -0700

Abstract

Through the integration of science, art, and technology, we present Multimedia Resolution Data Artistic Educational Tool (MRDAET), an interactive installation designed to support the visualization and understanding of molecular reactions. The installation focuses on ATP synthesis and the electron transport chain—core biochemical processes that are often difficult to grasp due to their complexity and abstract nature. Inspired by David Goodsell's fusion of scientific accuracy and artistic expression, MRDAET employs physical 3D models derived from structural data in the Protein Data Bank, augmented with projection mapping and enhanced with illustrations. The installation encourages users to explore molecular processes through layered interactivity that combines tangible models with animated visual overlays. MRDAET was evaluated at an art and technology event, where user feedback indicated that the installation was both enjoyable and perceived as helpful in education. Participants reported improved understanding of the presented biochemical concepts and expressed interest in increased interactivity, particularly through Mixed Reality integration connecting physical models and dynamic animations. While individual and combined visual modalities have been shown to be effective in science education, immersive interactive installations of this type remain underexplored in this context. This paper presents MRDAET as the third iteration following prior designs, offering a science-driven educational approach that warrants further study and application to additional scientific areas.

Description of the rate‐limiting hydrogen tunneling mechanism for plant 9‐ and 13‐lipoxygenases

Tue, 02 Jun 2026 03:13:41 -0700

Abstract

Soybean lipoxygenase (GmLOX1), a plant 13-LOX, has long been considered a model protein for non-trivial, quantum hydrogen tunneling in enzyme catalysis. Hydrogen tunneling mechanisms have also been confirmed for LOXs across mammalian, fungal, and bacterial kingdoms. Notably, GmLOX1 exhibits a low activation energy (E_a) for catalysis relative to characterized non-plant LOXs. In the present study, we examined three additional plant isozymes, including representatives of both 9- and 13-LOXs. All plant LOXs show large primary deuterium kinetic isotope effects associated with rate-limiting hydrogen tunneling during cleavage of a carbon-hydrogen bond on substrate linoleic acid. The monomeric plant 9-LOXs studied herein uniquely display kinetic cooperativity in their steady-state kinetics, while GmLOX1 does not. These data implicate fatty acid-dependent structural regulation of plant 9-LOX catalysis in a similar manner to mammalian LOXs. In lieu of experimental structures of these plant LOXs with substrate, we predicted representative 9- and 13-LOX isoforms in AlphaFold3 (AF3) with fatty acids and compared them with their crystal structures to better predict modes of substrate acquisition that could support the alternative kinetic models. Further, we find that two 9-LOXs have larger E_a values relative to GmLOX1. To understand this property, we also present a visualization of LOX evolutionary conservation across the plant kingdom. The data provide deeper insights into the reaction mechanisms of plant LOXs in the context of the activation barriers for catalysis and the relationships to the thermal activation of hydrogen tunneling.

In memoriam: Neville Robert Kallenbach (1938–2026)

Paramjit S. Arora, S. Walter Englander, George D. Rose — Tue, 02 Jun 2026 03:12:39 -0700

Protein Science, Volume 35, Issue 7, July 2026.

Interdomain linkers tune the energy dissipation of immunoglobulin domains in titin

Mon, 01 Jun 2026 21:13:04 -0700

Abstract

Mechanical forces are central to biological function, with mechanosensitive multidomain proteins (MDPs) such as titin translating strain into adaptive responses. While damping is well recognized at the tissue level in muscles, how it emerges from a multidomain protein connected by interdomain linkers (IDLs) has remained unclear. Here, using magnetic tweezers, we probe engineered titin I27 dimers connected by three distinct synthetic linkers (RS, GG, and GGGSGGGG) under both equilibrium constant force clamp and far-from-equilibrium oscillatory force pulse protocols. We show that folded domains exhibit unfolding along with deformations under load and that IDLs act as molecular regulators of damping. Rigid linkers promote stepwise, cooperative unfolding and faster energy dissipation, showing overdamped response, whereas flexible linkers favor gradual, viscoelastic transitions with energy-conserving underdamped behavior. Furthermore, increased domain stability correlates with enhanced damping capacity. Our work provides experimental evidence of single protein-level damping, revealing how nature encodes long-term durability into its molecular machines.

AcrIIA8 is a putative phage structural protein of the HTJ2 family that does not inhibit Streptococcus pyogenes Cas9

So Young An, Iktae Kim, Sung‐Hyun Hong, Eun‐Hee Kim, Jeong‐Yong Suh — Mon, 01 Jun 2026 04:16:58 -0700

Abstract

Anti-CRISPR (Acr) proteins are phage-encoded anti-defense factors that suppress CRISPR-Cas immunity in bacteria. AcrIIA8 was previously identified as an inhibitor of Streptococcus pyogenes Cas9 (SpyCas9) through functional assays of metagenomic libraries. Here, we report that AcrIIA8 does not inhibit SpyCas9 in biochemical assays under a range of buffer conditions and temperatures. The solution structure and dynamics of AcrIIA8 reveal a six-stranded β-barrel fold with flexible β1–β2 and β2–β3 loops, characteristic of phage virion-assembly proteins. In addition, genomic context analysis places AcrIIA8 and its homologs within conserved prophage morphogenetic regions at the position expected for type II head-tail joining (HTJ2) proteins. We further detected no interaction between AcrIIA8 and SpyCas9 in NMR titration experiments, suggesting that they do not specifically associate. Taken together, these findings argue against assigning AcrIIA8 as a SpyCas9 inhibitor and instead support its annotation as a putative phage structural protein of the HTJ2 family.

Unveiling catalytic potential and the native role of PETase from Streptomyces sp.

Mon, 01 Jun 2026 03:50:25 -0700

Abstract

Polyethylene terephthalate (PET) is one of the most widely produced thermoplastics, valued for its durability and low cost, yet it contributes significantly to global environmental pollution. The discovery of PET-degrading enzymes in Actinobacteria inspired a bioinformatics search for novel PET hydrolases in Streptomyces species from Chile's Atacama Desert. We selected PETS26b for detailed characterization. Genomic context revealed conserved CAZymes adjacent to PETS26b. Biochemical characterization showed optimal activity at 28°C and pH 8.0. PETS26b exhibited broad substrate specificity toward p-nitrophenyl esters, with superior catalytic efficiency toward p-nitrophenyl octanoate (kcat/Km = 2.92 × 10⁶ M⁻¹ s⁻¹) relative to other reported esterases. The enzyme also hydrolyzed PET powder and bis(2-hydroxyethyl) terephthalate (BHET), and deacetylated glucose pentaacetate, consistent with generalist esterase activity. Overall, docking analyses revealed favorable docking scores for 4-nitrophenyl esters and PET-related substrates within the active site, with predicted docking scores showing good agreement with experimental kinetic data. Moreover, PETS26B accommodated variably acetylated carbohydrate oligosaccharides, displaying particularly favorable binding scores for highly acetylated substrates. These findings highlighted the versatile roles of PET-degrading enzymes in the degradation of plant biomass esters.

Cover Image

Mon, 01 Jun 2026 02:37:06 -0700

Water forms cohesive interactions (hydrogen bonds) in the liquid and solid state. The density of hydrogen bonds in water is greater than for any other known substance. For details, see: Juliana DiGiacomo et al., Protein Science, 2026. https://doi.org/10.1002/pro.70532

Issue information

Mon, 01 Jun 2026 02:35:03 -0700

Protein Science, Volume 35, Issue 7, July 2026.