Biology subjects

Adoni, K. R.

Publications and source records attributed to Adoni, K. R..

4 recordsLinked to original sources

bioRxiv2026

Cysteine Glutathionylation as a Global Dynamic Regulator of Protein Active Site Accessibility and Protein Complex Formation

Protein-glutathionylation is traditionally viewed as a protective mechanism that shields cysteine-residues from irreversible oxidative damage. Its broader functional roles remain poorly understood, in part due to technical limitations in detecting this modification at scale. Here, we develop and leverage a new mass-spectrometry approach that preserves protein-glutathionylation, thereby revealing its widespread distribution across the proteome in cell models, worms, mice and human cardiac tissues. In all cases, we find that glutathionylation sites are enriched at both protein-protein interfaces and protein-active sites, and are highly conserved across species. We find that glutathionylation is dynamically redistributed in response to environmental challenges, thereby driving remodelling of cellular protein-protein interaction (PPI) networks, and access to protein active sites, with functional and phenotypic consequences. Finally, we show that glutathionylation accumulates on key cardiac-sarcomeric proteins in aged-mice and human cardiomyopathy biopsies, revealing its role in cardiovascular dysfunction. These findings reposition glutathionylation as a crucial regulatory PTM, akin to phosphorylation, that orchestrates adaptive cellular responses. This work redefines the role of glutathionylation, with broad implications for cell biology and disease.

molecular biology↗

bioRxiv2025

Enhanced Proteomics Analysis with a Novel Recombinant Chymotrypsin Analogue Engineered for High Cleavage Specificity

Chymotrypsin is widely used in shotgun proteomics owing to its orthogonal cleavage specificity relative to trypsin, which enhances sequence coverage of hydrophobic protein regions. However, commercial preparations often display variable cleavage specificity, trypsin contamination, and elevated missed-cleavage rates, which can collectively reduce proteome coverage and data reproducibility. To address these limitations, we present a novel recombinant chymotrypsin (rChymoSelect) engineered for improved cleavage specificity and robustness in proteomics workflows. Benchmarking against standard bovine chymotrypsin revealed 97 % C-terminal cleavages after tyrosine (Y), phenylalanine (F), and leucine (L) for rChymoSelect, compared with 72 % for the standard enzyme. This enhanced cleavage specificity reduced missed cleavages and increased peptide-spectrum matches across charge states. Across 3,638 identified proteins, rChymoSelect yielded 22.2 % unique identifications compared with 8.2 % for standard chymotrypsin, while maintaining similar peptide length, m/z, and hydrophobicity distributions. Notably, rChymoSelect showed enriched recovery of mitochondrial proteins, consistent with its improved digestion of hydrophobic targets. The enzyme remained active in up to 6 M urea and achieved near-maximal proteome coverage within 2 hours (only a 2.4 % gain after overnight digestion). Integration with data-independent acquisition (DIA) increased total protein identifications from approximately 2,200 (DDA) to 3,200 (DIA), a 45 % gain, with rChymoSelect outperforming standard chymotrypsin by 16.6-22.4 % in peptide-spectrum matches and 4.6-6.2 % in protein identifications. These results establish rChymoSelect as an advanced tool with improved cleavage specificity that reduces analytical complexity and enhances the reliability of proteomic analysis, while expanding chymotryptic digestion to hydrophobic and high-denaturant proteomics applications.

biochemistry↗

bioRxiv2025

NanoBondy reacting through NeissLock anhydride allows covalent immune cell decoration

Cell-surface conjugation has enormous therapeutic and research potential. Existing technologies for cell-surface modification are usually reversible, non-specific, or rely on genetic editing of target cells. Here we present the NanoBondy, a nanobody modified for covalent ligation to an endogenous protein target at the cell-surface. The NanoBondy utilizes the 20 naturally occurring amino acids, harnessing NeissLock chemistry engineered from Neisseria meningitidis. We evaluated binding and specificity of a panel of nanobodies to CD45, a long-lived surface marker of nucleated hematopoietic cells. We demonstrated conversion of existing nanobodies to covalently reacting NanoBondies using a disulfide clamp to position the self-processing module of FrpA close to the nanobody antigen-binding site. Addition of calcium induces anhydride formation at the NanoBondy C-terminus and proximity-directed ligation to surface amines on CD45. We optimized NanoBondy reaction by fine-tuning linkers and disulfide clamp sites to modulate anhydride positioning. Tandem mass spectrometry mapped reaction sites between the NanoBondy and CD45. NanoBondy ligation was robust to buffer, pH and temperature and was detected within 2 minutes. We established reaction specificity of NanoBondies to endogenous CD45 at the surface of NK cells and T cells. NanoBondy technology provides a modular approach for targeted, inducible and covalent cell-surface modification of immune cells.

synthetic biology↗

bioRxiv2023

Modeling flexible protein structure with AlphaFold2 and cross-linking mass spectrometry

We propose a pipeline that combines AlphaFold2 (AF2) and crosslinking mass spectrometry (XL-MS) to model the structure of proteins with multiple conformations. The pipeline consists of two main steps: ensemble generation using AF2, and conformer selection using XL-MS data. For conformer selection, we developed two scores - the monolink probability score (MP) and the crosslink probability score (XLP), both of which are based on residue depth. We benchmarked MP and XLP on a large dataset of decoy protein structures, and showed that our scores outperform previously developed scores. We then tested our methodology on three proteins having an open and closed conformation in the Protein Data Bank: Complement component 3 (C3), luciferase, and glutamine-binding periplasmic protein (QBP), first generating ensembles using AF2, which were then screened for the open and closed conformations using experimental XL-MS data. In five out of six cases, the most accurate model within the AF2 ensembles - or a conformation within 1 [A] of this model - was identified using crosslinks, as assessed through the XLP score. In the remaining case, only the monolinks (assessed through the MP score) successfully identified the open conformation of QBP. This serves as a compelling proof-of-concept for the effectiveness of monolinks. In contrast, the AF2 assessment score (pTM) was only able to identify the most accurate conformation in two out of six cases. Our results highlight the complementarity of AF2 with experimental methods like XL-MS, with the MP and XLP scores providing reliable metrics to assess the quality of the predicted models.

biochemistry↗