1. Differential scanning calorimetry of proteins and Zimm–Bragg model in waterKnarik Yeritsyan, Artem Badasyan, 2019, izvirni znanstveni članek Opis: Differential Scanning Calorimetry (DSC) is a regular and powerful tool to measure the specific heat profile of various materials. Hydrogen bonds play a crucial role in stabilizing the three-dimensional structure of proteins. Naturally, information about the strength of hydrogen bonds is contained in the measured DSC profiles. Despite its obvious importance, there is no approach that would allow the extraction of such information from the heat capacity measurements. In order to connect the measured profile to microscopic properties of a polypeptide chain, a proper model is required to fit. Using recent advances in the Zimm–Bragg (ZB) theory of protein folding in water, we propose a new and efficient algorithm to process the DSC experimental data and to extract the H-bonding energy among other relevant constants. Thus, for the randomly picked set of 33 proteins, we have found a quite narrow distribution of hydrogen bonding energies from 1 to 8 kJ/mol with the average energy of intra-protein hydrogen bonds kJ/mol and the average energy of water–protein bonds as kJ/mol. This is an important illustration of a tiny disbalance between the water–protein and intraprotein hydrogen bonds. Fitted values of the nucleation parameter belong to the range from 0.001 to 0.01, as expected. The reported method can be considered as complementary to the classical two-state approach and together with other parameters provides the protein–water and intraprotein H-bonding energies, not accessible within the two-state paradigm. Ključne besede: protein folding, differential scanning calorimetry, heat capacity, two-state model, Hawley model Objavljeno v RUNG: 26.08.2024; Ogledov: 594; Prenosov: 4 Celotno besedilo (645,94 KB) Gradivo ima več datotek! Več... |
2. Functional characterisation of surfactant protein A as a novel prophylactic means against oncogenic HPV infectionsSinead Carse, Tim Reid, Jens Madsen, Howard Clark, Artur Kirjakulov, Martina Bergant Marušič, Georgia Schäfer, 2024, izvirni znanstveni članek Opis: Human papillomavirus (HPV) infection poses a significant health challenge, particularly in low- and middle-income countries (LMIC), where limited healthcare access and awareness hinder vaccine accessibility. To identify alternative HPV targeting interventions, we previously reported on surfactant protein A (SP-A) as a novel molecule capable of recognising HPV16 pseudovirions (HPV16-PsVs) and reducing infection in a murine cervicovaginal HPV challenge model. Building on these findings, our current study aimed to assess SP-A’s suitability as a broad-spectrum HPV-targeting molecule and its impact on innate immune responses. We demonstrate SP-A’s ability to agglutinate and opsonise multiple oncogenic HPV-PsVs types, enhancing their uptake and clearance by RAW264.7 murine macrophages and THP-1 human-derived immune cells. The SP-A opsonisation of HPV not only led to increased lysosomal accumulation in macrophages and HaCaT keratinocytes but also resulted in a decreased infection of HaCaT cells, which was further decreased when co-cultured with innate immune cells. An analysis of human innate immune cell cytokine profiles revealed a significant inflammatory response upon SP-A exposure, potentially contributing to the overall inhibition of HPV infection. These results highlight the multi-layered impact of SP-A on HPV, innate immune cells and keratinocytes and lay the basis for the development of alternative prophylactic interventions against diverse HPV types. Ključne besede: surfactant protein A, SP-A, human papillomavirus, HPV, pseudovirus, cervical cancer, HPV prophylactic, low- and middle-income countries, LMIC Objavljeno v RUNG: 22.07.2024; Ogledov: 779; Prenosov: 7 Celotno besedilo (6,10 MB) Gradivo ima več datotek! Več... |
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4. Native agarose gels and contact blotting as means to optimize the protocols for the formation of antigen-ligand complexesClaudia D'Ercole, Ario De Marco, 2024, objavljeni povzetek znanstvenega prispevka na konferenci Opis: Background. Protein complexes provide valuable biological information but can be difficult to handle [1]. Therefore, technical advancements designed to improve their manipulation are al-ways useful.
Methods. We investigated the opportunity to exploit native agarose gels and the contact bot method for the transfer of native proteins to membranes as means for optimizing the conditions for obtaining stable complexes [2]. As a simple model of protein-protein interactions, an anti-gen-ligand complex was used in which both proteins were fused to reporters.
Results. At each step, it was possible to visualize both the antigen, fused to a fluorescent pro-tein, and the ligand, fused to a monomeric ascorbate peroxidase (APEX) and, as such, a way to tune the protocol. The conditions for the complex formation were adapted by modifying the buffer conditions, the concentration of the proteins and of the cross-linkers.
Conclusions. The procedure is rapid, inexpensive, and the several detection opportunities al-lows for both the monitoring of complex stability and the preservation of the functionality of its components, which is critical for understanding their biomedical implications and supporting drug discovery. The overall protocol represents a handy alternative to gel filtration, uses very standard and ubiquitous equipment, and can be implemented rapidly and without specific train-ing.
References:
[1] O. Puig, F. Caspary, G. Rigaut, B. Rutz, E. Bouveret, E. Bragado-Nilsson, M. Wilm, B. Séraphin. The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods. (San Diego, Calif.), 24(3), 218–229
[2] C. Sakuma, M. Nakagawa, Y. Tomioka, T. Maruyama, K. Entzminger, J.K. Fleming, T. Shibata, Y. Kurosawa, C.J. Okumura, T. Arakawa, T. Akuta. Western blotting of native proteins from agarose gels. Biotechniques. 2022 May;72(5):207-218 Ključne besede: protein complexes, contact blotting, native agarose gels, protein interaction Objavljeno v RUNG: 17.06.2024; Ogledov: 826; Prenosov: 1 Povezava na datoteko Gradivo ima več datotek! Več... |
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6. Instruct-ERIC network : biophysical characterization of antigen-nanobody complexesClaudia D'Ercole, 2024, objavljeni povzetek znanstvenega prispevka na konferenci Opis: 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. Ključne besede: nanobody, ascorbate peroxidase, plant stress, protein complex, biophysical methodologies Objavljeno v RUNG: 31.05.2024; Ogledov: 1017; Prenosov: 0 Gradivo ima več datotek! Več... |
7. Differential scanning calorimetry of proteins and the two-state model : comparison of two formulasKnarik Yeritsyan, Artem Badasyan, 2024, izvirni znanstveni članek Opis: 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. Ključne besede: protein folding, differential scanning calorimetry, heat capacity, two-state model, Hawley model Objavljeno v RUNG: 21.05.2024; Ogledov: 1103; Prenosov: 7 Celotno besedilo (380,96 KB) Gradivo ima več datotek! Več... |
8. A practical guide for the quality evaluation of fluobodies/chromobodiesUrša Štrancar, Claudia D'Ercole, Lucia Cikatricisová, Mirna Nakić, Matteo De March, Ario De Marco, 2024, izvirni znanstveni članek Ključne besede: fluorescent proteins, protein stability, protein degradation Objavljeno v RUNG: 16.05.2024; Ogledov: 942; Prenosov: 6 Celotno besedilo (667,85 KB) Gradivo ima več datotek! Več... |
9. A concise guide to choosing suitable gene expression systems for recombinant protein productionAnja Schütz, Frank Bernhard, Nick Berrow, Johannes F. Buyel, Frederico Ferreira-da-Silva, Jurgen Haustraete, Joop Van den Heuvel, Jan-Erik Hoffmann, Ario De Marco, Yoav Peleg, 2023, izvirni znanstveni članek Ključne besede: recombinant protein, protein expression options, post-translational modifications Objavljeno v RUNG: 06.11.2023; Ogledov: 1817; Prenosov: 6 Celotno besedilo (3,14 MB) Gradivo ima več datotek! Več... |
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