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Processing CD and DSC data on protein folding with Zimm-Bragg model in water
Artem Badasyan, Knarik Yeritsyan, 2024, published scientific conference contribution abstract

Abstract: Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC) data are processed with a novel model incorporating water effects and inter-/intra-molecular hydrogen bonding energies to better fit experimental data on protein folding as compared to the two-state approach.
Keywords: protein folding, circular dichroism, differential scanning calorimetry, water-protein interactions
Published in RUNG: 10.06.2024; Views: 112; Downloads: 0
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Instruct-ERIC network : biophysical characterization of antigen-nanobody complexes
Claudia D'Ercole, 2024, published scientific conference contribution abstract

Abstract: Forest environments are exposed to multiple stressful factors of both abiotic and biotic nature which may lead to their massive decline [1]. Understanding the molecular mechanisms of specific stress conditions and monitoring the fluctuations of reliable forest plant biomarkers with affordable methods would be instrumental for assessing stress levels over the time. Ascorbate peroxidase (APX) represents a suitable plant biomarker. APX is a hydrogen peroxide-scavenging enzyme the critical role of which has been described in several plants, both herbaceous and woody. Its activity generally increases under oxidative stress during which its peroxide detoxifying function is part of the wider ascorbate-glutathione cycle [2]. The development of reagents to detect such fluctuations would help the evaluation of plant physiological conditions. In this study, nanobodies (Nbs) targeting APX have been identified. Nbs correspond to the variable domain of heavy chain-only antibodies derived from camelids. They are small (15 kDa), stable, and can be easily produced in bacteria fused to different protein tags according to the downstream applications [3]. After their isolation by biopanning against soluble APX, they have been produced and underwent a biophysical characterization in combination with their antigen (APX-Nb complex) to identify the best binders in terms of stability and affinity. The protein complex characterization was supported by Instruct-ERIC and mainly performed at the BIOCEV institute of Prague. Data from Mass Photometry and Dynamic Light scattering evidenced the formation of the protein complexes, whereas the preliminary data of Hydrogen-Deuterium Exchange Mass Spectrometry, performed with the aim of identifying the residues involved in the paratope/epitope interface, were insufficient to clarify the issue and rather suggested that the interaction has low affinity. This indication was then confirmed by ELISA assay. The combination of multiple methods allowed a comprehensive sample characterization which will require further structural analyses to provide a complete picture of the APX-Nb complex. [1] G. Marie. B. C. M. B. C. Walters, “Forest decline and tree mortality in a southeastern Ohio oak-hickory forest,” Ohio Journal of Science , vol. 97, 1997. [2] O. Chew, J. Whelan, and A. H. Millar, “Molecular Definition of the Ascorbate-Glutathione Cycle in Arabidopsis Mitochondria Reveals Dual Targeting of Antioxidant Defenses in Plants,” Journal of Biological Chemistry, vol. 278, no. 47, 2003, doi: 10.1074/jbc.M307525200. [3] S. Muyldermans, “A guide to: generation and design of nanobodies,” FEBS J, vol. 288, no. 7, pp. 2084–2102, Apr. 2021, doi: 10.1111/febs.15515.
Keywords: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies
Published in RUNG: 31.05.2024; Views: 307; Downloads: 0
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Differential scanning calorimetry of proteins and the two-state model : comparison of two formulas
Knarik Yeritsyan, Artem Badasyan, 2024, original scientific article

Abstract: Differential Scanning Calorimetry (DSC) is a regular and powerful tool to measure the specific heat profile of various materials. In order to connect the measured profile to the properties of a particular protein, a model is required to fit. We discuss here the application of an exact two-state formula with its approximation and process the DSC experimental data on protein folding in water. The approximate formula relies on the smallness of the transition interval, which is different for each protein. With an example of the set of 33 different proteins, we show the practical validity of the approximation and the equivalence of exact and approximate two-state formulas for processing DSC data.
Keywords: protein folding, differential scanning calorimetry, heat capacity, two-state model, Hawley model
Published in RUNG: 21.05.2024; Views: 344; Downloads: 3
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A practical guide for the quality evaluation of fluobodies/chromobodies
Urša Štrancar, Claudia D'Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March, Ario De Marco, 2024, original scientific article

Keywords: fluorescent proteins, protein stability, protein degradation
Published in RUNG: 16.05.2024; Views: 250; Downloads: 2
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Protein quality assessment: why & how
Ario De Marco, 2023, unpublished conference contribution

Keywords: recombinant protein, protein quality control, protein aggregates
Published in RUNG: 02.11.2023; Views: 965; Downloads: 0
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Modelling water for calorimetry of proteins
Knarik Yeritsyan, Artem Badasyan, 2023, published scientific conference contribution abstract

Abstract: Differential Scanning Calorimetry (DSC) is a powerful technique used to study the thermal stability and unfolding of proteins. DSC provides the excess heat capacity profile and is used to study the thermodynamics of a given protein. By fitting DSC data to the model, researchers can obtain valuable information about the thermodynamics of protein folding and unfolding, which can help them better understand protein structure, stability, and function. Based on Hamiltonian representation of ZB model and using the solvent effects we derived an expression for heat capacity in proteins and successfuly fit it to experimental data. As we show, our model provides a better fit to experimental data, as compared to the 2-state model. The model we propose takes into account also water effects and we show that it fits better to experimental data giving inter- and intra-molecular H-bonding energies instead of reporting only one total enthalpy.
Keywords: Zimm-Bragg model, water model, helix-coil transition, protein folding, differential scanning calorimetry
Published in RUNG: 18.10.2023; Views: 942; Downloads: 0
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Spin model of water and post-processing of protein folding experiments
Artem Badasyan, Knarik Yeritsyan, 2023, published scientific conference contribution abstract

Abstract: The presence of several distinct minima in nearest-neighbor potentials in polymers makes it possible to describe polymer conformations in terms of discrete isomeric states, naturally leading to spin language. Using this general approach, a decade ago we have suggested the Hamiltonian formulation for the Zimm and Bragg model of protein conformations [1,2]. Later we have augmented the model by an oversimplified spin model for water, resulting in both cold and hot denaturations [3]. We construct the Statistical Mechanics for the model and get access to its Thermodynamics. Resulting order parameter and specific heat expressions are successfully fit to available experimental data [4]. Thanks to solid and traceable theoretical foundations, the procedure provides better quality fits as compared to the state-of-the-art two-state model, routinely used to process protein folding experiments.
Keywords: spin model, water, protein folding
Published in RUNG: 04.09.2023; Views: 998; Downloads: 7
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