Biology subjects

Nowak, J. S.

Publications and source records attributed to Nowak, J. S..

6 recordsLinked to original sources

bioRxiv2026

The Thermodynamics of Biomolecular CO2 Capture:Disentangling Equilibria in Amino-Acid-based Systems

Amino acids and peptides are promising building blocks for aqueous biomolecular CO2 capture systems, yet the coupled thermodynamics governing carbamate formation, proton transfer, carbonate speciation, and hydration remain difficult to resolve experimentally. Here, we establish isothermal titration calorimetry (ITC) as a quantitative platform for characterizing these coupled processes by integrating calorimetry with pH titrations, NMR spectroscopy, and a mechanistic thermodynamic model. Using L-lysine, L-arginine, and a series of Lys- and Arg-containing peptides, global fitting of ITC thermograms yielded thermodynamic parameters describing protonation and carbamate formation that accurately reproduced independent pH titrations and NMR-derived speciation. The analysis revealed that the characteristic biphasic calorimetric response originates from the coupled carbonate-amine equilibrium network and buffer collapse rather than carbamate saturation. Lys formed -, {varepsilon}-, and ,{varepsilon}-dicarbamates and exhibited more favorable apparent carbamate thermodynamics than Arg with the {varepsilon}-carbamate lying among the most favorable carbamate-forming amine sites reported for aqueous amines. Model-guided exploration of the fitted thermodynamic landscape further demonstrated that maximizing total CO2 retention, amine-mediated capture, and carbamate formation are distinct optimization problems governed by different combinations of pH, temperature, and CO2 loading. Extension to systematically spaced Lys-containing peptides showed that inter-amine separation alone does not control carbamate stability, highlighting the dominant role of the local thermodynamic environment in biomolecular CO2 capture. This work establishes ITC as a powerful experimental approach for extracting CO2-amine thermodynamics and provides a predictive framework for the rational design and optimization of amino acid-, peptide-, and protein-based carbon capture systems.

biochemistry↗

bioRxiv2026

Global analysis of thermal and chemical denaturation using CheMelt: Thermodynamic dissection of highly thermostable de novo designed proteins

De novo protein design often produces thermostable proteins that denature above 100 {degrees}C, which complicates the analysis of their stability. Thermostable proteins can be unfolded by combined chemical and thermal denaturation followed by global analysis of multiple melting curves. Here, we have developed CheMelt, a new online tool for global analysis of unfolding data via an intuitive graphical user interface. We use nanoscale differential scanning fluorimetry followed by CheMelt data analysis to dissect the combined thermal and chemical denaturation of thirty-five de novo designed protein binders. Thirteen present sufficient fluorescence changes to extract thermodynamic parameters of unfolding. These de novo designed proteins have systematically lower {Delta}Cp and m-values than comparable natural proteins. We show that a high thermostability of a designed protein does not necessarily imply a high equilibrium stability, and demonstrate the potential of CheMelt in dissecting thermodynamic properties for protein design and engineering. CheMelt can be accessed at https://spc.embl-hamburg.de/app/chemelt

biophysics↗

bioRxiv2025

Screening de novo designed protein binders in unpurified lysate using flow induced dispersion analysis

Computational protein design can create binders against targets of interest, but identifying binders with sufficient affinity still requires biochemical screening of many designs. In this work, we test flow-induced dispersion analysis (FIDA) as a method for screening binders in a time and cost-effective manner. FIDA uses Taylor dispersion analysis to determine the hydrodynamic radius of fluorescently labelled biomolecules and their complexes. Here, we use FIDA to assess the binding of RFdiffusion-designed protein binders against the small helical peptide ALFA-tag and the GK domain of PSD-95. Successful binders can be identified in a single measurement using heat-treated bacterial lysates allowing rapid identification of binders with high affinity, and thermostability. Subsequent titration experiments show micromolar affinities for ALFA-tag binders and nanomolar affinities for GK domain binders. The lack of immobilization, the minimal sample volume, and the compatibility with complex biological samples, positions FIDA as a valuable tool for the screening and characterization of computationally generated protein binders.

biophysics↗

bioRxiv2023

Specific inhibition of α-synuclein oligomer generation and toxicity by the chaperone domain Bri2 BRICHOS

Understanding the molecular mechanisms of neurodegenerative diseases and finding efficient treatments have been major priorities for research and society, yet new therapeutic approaches remain essential to face the socio-economic burden caused by these devastating diseases. Protein misfolding and aggregation are involved in several neurodegenerative disorders, such as -synuclein (Syn) implicated in Parkinsons disease. Elucidating the microscopic nucleation mechanisms has opened new opportunities to develop therapeutics against toxic mechanisms and species. Here, we show that naturally occurring molecular chaperones, represented by the anti-amyloid Bri2 BRICHOS domain, can be used to target Syn-associated nucleation processes and structural species related to neurotoxicity. Our findings revealed that BRICHOS predominately suppresses the formation of new nucleation units on the fibrils surface (secondary nucleation), in addition to fibril-end elongation. This mechanism implies a drastic decrease of the oligomer generation rate. Besides targeting secondary nucleation sites on the fibril surface, BRICHOS directly binds to oligomeric Syn species. Further, using ex vivo experiments, BRICHOS effectively diminishes Syn fibril-related toxicity to hippocampal electrophysiology. Our studies show that molecular chaperones can be utilized as tools to target molecular processes and structural species related to Syn neurotoxicity and have the potential as protein-based treatments against neurodegenerative disorders.

biophysics↗

bioRxiv2020

Quantitating denaturation by formic acid: Imperfect repeats are essential to the stability of the functional amyloid protein FapC

Bacterial functional amyloids are evolutionarily optimized to aggregate to help them fulfil their biological functions, e.g. to provide mechanical stability to biofilm. Amyloid is formed in Pseudomonas sp. by the protein FapC which contains 3 imperfect repeats connected by long linkers. Stepwise removal of these repeats slows down aggregation and increases the propensity of amyloids to fragment during the fibrillation process, but how these mechanistic properties link to fibril stability is unclear. Here we address this question. The extreme robustness of functional amyloid makes them resistant to conventional chemical denaturants, but they dissolve in formic acid (FA) at high concentrations. To quantify this, we first measured the denaturing potency of FA using 3 small acid-resistant proteins (S6, lysozyme and ubiquitin). This revealed a linear relationship between [FA] and the free energy of unfolding with a slope of mFA, as well as a robust correlation between protein residue size and mFA. We then measured the solubilisation of fibrils formed from different FapC variants (with varying number of repeats) as a function of [FA]. The resulting mFA values revealed a decline in the number of residues driving amyloid formation when at least 2 repeats were deleted. The midpoint of denaturation declined monotonically with progressive removal of repeats and correlated with solubility in SDS. Complete removal of all repeats led to fibrils which were solubilized at FA concentrations 2-3 orders of magnitude lower than the repeat-containing variants, showing that at least one imperfect repeat is required for the stability of functional amyloid.

biophysics↗

bioRxiv2017

RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination

TRIM25 is a novel RNA-binding protein and a member of the Tripartite Motif (TRIM) family of E3 ubiquitin ligases, which plays a pivotal role in the innate immune response. Almost nothing is known about its RNA-related roles in cell biology. Furthermore, its RNA-binding domain has not been characterized. Here, we reveal that RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain, which we postulate to be a novel RNA-binding domain. Using CLIP-seq and SILAC-based co-immunoprecipitation assays, we uncover TRIM25s endogenous RNA targets and protein binding partners. Finally, we show that the RNA-binding activity of TRIM25 is important for its ubiquitin ligase function. These results reveal new insights into the molecular roles and characteristics of RNA-binding E3 ubiquitin ligases and demonstrate that RNA could be an essential factor for their biological functions.

molecular biology↗