| 1. |
- Caporaletti, Francesca, 1990-, et al.
(författare)
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Small-angle X-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding model.
- 2023
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 122:2, s. 408-418
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Tidskriftsartikel (refereegranskat)abstract
- In this work, we used Small-angle X-ray and neutron scattering (SAS) to reveal the shape of the protein-DNA complex of the Pseudomonas aeruginosa (P.aeruginosa) transcriptional regulator MexR, a member of the MarR family, when bound to one of its native DNA binding sites. Several MarR-like proteins, including MexR, repress the expression of efflux pump proteins by binding to DNA on regulatory sites overlapping with promoter regions. When expressed, efflux-proteins self-assemble to form multiprotein complexes and actively expel highly toxic compounds out of the host organism. The mutational pressure on efflux-regulating MarR family proteins is high since deficient DNA binding leads to constitutive expression of efflux pumps and thereby supports acquired multidrug resistance. Understanding the functional outcome of such mutations and their effects on DNA binding has been hampered by the scarcity of structural and dynamic characterisation of both free and DNA-bound MarR proteins. Here, we show how combined neutron and X-ray small-angle scattering (SAS) of both states in solution support a conformational selection model that enhances MexR asymmetry in binding to one of its promoter-overlapping DNA binding sites.
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| 2. |
- Pietras, Zuzanna, 1993-
(författare)
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Small angle scattering as a tool to study protein structure and interactions
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis uses small angle X-ray and neutron scattering (SAXS/SANS) to gain structural and functional insight into the molecular regulation of critical life processes in prokaryotic and eukaryotic species. The presented studies highlight the strength of combining low-resolution structure determination with biophysical and in silico modelling methods to extensively characterize proteins and their interactions. DNA-binding: MexR protein belongs to the family of bacterial transcription regulators and control the expression of multidrug efflux pumps in Pseudomonas Aeruginosa by binding to a DNA region of the operator. SAXS/SANS data supported by MD (Molecular Dynamics) simulations demonstrated that the MexR dimer in solution undergoes a DNA-binding conformational selection mechanism. To gain a better understanding about the system, a low-resolution structural model was resolved in order to assess protein binding to the entire operator region comprising of two closely located DNA recognition sites. The study demonstrates that the use of scattering techniques to investigate similar systems is straightforward and provides knowledge of relevance for clinical understanding and future drug design. Viral host factors: Picornaviruses represent a large family of small RNA viruses that are responsible for a range of diseases in humans and animals. Recently a non-essential human phospholipase PLAAT3 was identified as a key host factor for some picornaviruses. Several picornaviruses representing different branches of the picornaviral phylogenetic tree contain a type of 2A protein in their genome that share a conserved H-box/NC motif with PLAAT3. To understand the role of these 2A proteins in the viral life cycle and to map their plasticity, high resolution techniques were complemented with SAXS to evaluate the structural rearrangements and flexibility. Ubiquitination: In eukaryotes, ubiquitination is a fundamental posttranslational modification, where a small protein ubiquitin is covalently attached to a target protein via sophisticated multienzyme process. SANS can be used to study this mechanism in solution by modular deuteration of ubiquitin complexes. To explore this possibility further, an E2 conjugating enzyme was attached to a deuterated ubiquitin via an isopeptide bond, and a neutron contrast variation experiment was performed. To investigate the flexibility of the E2~Ub conjugate, a multi-state modelling approach was employed to sample its conformational landscape. SANS methods in protein science: A final methods paper outlines and details the experimental requirements, procedures and pre-studies that need to be considered to optimise a successful experimental approach for SANS with contrast variation on biomolecular complexes and assemblies in solution.
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| 3. |
- Wehlin, Anna, 1994-
(författare)
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Structural and Functional Studies on Evolutionary Repurposing of H-box/NC-proteins : From Host Factor to Virus Protein
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Picornaviridae are a large family of biomedically important viruses causing diseases such as the common cold, hepatitis A and polio in humans and foot-and-mouth disease in cattle. These diseases have great impact on people’s everyday life and cause economical losses all around the world. To date, no antiviral treatments are available. In attempts to identify potential drug targets for novel antiviral therapies, a human protein was identified as a common host factor for several enteroviruses, a genus within the picornavirus family. This host factor, PLAAT3, facilitates genome transfer from the virus particle into the cytoplasm early in the viral lifecycle prior to virus clearance by autophagy. PLAAT3 is part of a human phospholipid-modifying enzyme family of five members, PLAAT1-5, which all have a conserved H-box/NC-motif forming the active site of these enzymes as well as a hydrophobic C-terminal region that is critical for enzymatic function. This H-box/ NC-motif is also found in the 2A locus of some picornaviruses, suggesting that these viruses might have acquired the protein through horizontal gene transfer to become independent of the human host factor.This thesis focuses on understanding the structural mechanism allowing picornavirus infection. Therefore, two members of the PLAAT-family were studied together with viral 2A proteins sharing the H-box/NC-motif.PLAAT3 was studied with the aim to elucidate its molecular mechanism underpinning its role as a host factor enabling genome transfer. PLAAT3 is composed of a globular N-terminal domain (NTD), whose structure has previously been determined, followed by a 30 amino acid long hydrophobic region (CTR). The catalytic site is located within the NTD, but the hydrophobic CTR is essential both for the catalytic activity as well as cellular localization of PLAAT3.PLAAT4 shares 50% sequence identity with PLAAT3 and exhibits a similar structure with a globular NTD followed by a hydrophobic tail. However, PLAAT4 shows a different activity pattern and displays enzymatic activity even in the absence of the CTR. By comparing the structural properties of PLAAT3 and PLAAT4 more can be understood of the structural characteristics enabling biological functions.The viral 2A proteins studied in this thesis originate from different picornavirus genera but all share the conserved H-box/NC-motif with the PLAAT-family. By investigating the structure and function of representative 2AH/NC proteins from different branches of the phylogenetic tree we aim to identify different steps of evolutionary repurposing to help us understand their role(s) in the viral lifecycle and determine the molecular mechanism allowing them to by-pass PLAAT3 as a host factor.
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| 4. |
- Ahmed, Niaz, et al.
(författare)
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Consensus statements and recommendations from the ESO-Karolinska Stroke Update Conference, Stockholm 11-13 November 2018.
- 2019
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Ingår i: European Stroke Journal. - : SAGE Publications. - 2396-9873 .- 2396-9881. ; 4:4, s. 307-317
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Tidskriftsartikel (refereegranskat)abstract
- The purpose of the European Stroke Organisation-Karolinska Stroke Update Conference is to provide updates on recent stroke therapy research and to give an opportunity for the participants to discuss how these results may be implemented into clinical routine. The meeting started 22 years ago as Karolinska Stroke Update, but since 2014 it is a joint conference with European Stroke Organisation. Importantly, it provides a platform for discussion on the European Stroke Organisation guidelines process and on recommendations to the European Stroke Organisation guidelines committee on specific topics. By this, it adds a direct influence from stroke professionals otherwise not involved in committees and work groups on the guideline procedure. The discussions at the conference may also inspire new guidelines when motivated. The topics raised at the meeting are selected by the scientific programme committee mainly based on recent important scientific publications. This year's European Stroke Organisation-Karolinska Stroke Update Meeting was held in Stockholm on 11-13 November 2018. There were 11 scientific sessions discussed in the meeting including two short sessions. Each session except the short sessions produced a consensus statement (Full version with background, issues, conclusions and references are published as web-material and at www.eso-karolinska.org and http://eso-stroke.org) and recommendations which were prepared by a writing committee consisting of session chair(s), scientific secretary and speakers. These statements were presented to the 250 participants of the meeting. In the open meeting, general participants commented on the consensus statement and recommendations and the final document were adjusted based on the discussion from the general participants Recommendations (grade of evidence) were graded according to the 1998 Karolinska Stroke Update meeting with regard to the strength of evidence. Grade A Evidence: Strong support from randomised controlled trials and statistical reviews (at least one randomised controlled trial plus one statistical review). Grade B Evidence: Support from randomised controlled trials and statistical reviews (one randomised controlled trial or one statistical review). Grade C Evidence: No reasonable support from randomised controlled trials, recommendations based on small randomised and/or non-randomised controlled trials evidence.
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| 5. |
- Caporaletti, Francesca, 1990-
(författare)
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MYC and MexR interactions with DNA : a Small Angle Scattering perspective
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-DNA complexes govern transcription, that is, the cellular mechanism that converts the information stored in the DNA into proteins. These complexes need to be highly dynamic to respond to external factors that regulate their functions in agreement with what the cells need at that time. Macromolecular X-ray crystallography is very useful for structural studies of large molecular assemblies, but its general application is limited by the difficulties in crystallising highly dynamic and transient complexes. Furthermore, crystal lattices constrain the macromolecular conformation and do not entirely reveal the conformational ensemble adopted by protein-DNA complexes in the solution.Small-Angle X-Ray Scattering (SAXS) and Small-Angle Neutron Scattering (SANS) are two complementary techniques known jointly as Small-angle Scattering (SAS). SAS is a powerful tool for analysing the shape and changes of molecules in solution in their native state. It is beneficial if the variability of conformation or disorder complements high-resolution methods such as NMR or crystallography. With SANS, we can explore non-crystallisable protein-DNA complexes in solution without restrictions of artificially symmetrised DNA and limitations of a protein sequence. Neutrons are well-suited probes for studying protein-DNA complexes for the capability of the neutrons to scatter common atoms in biomolecules differentially and can thereby distinguish between hydrogen and deuterium. Together with varying the solvent deuterium ratio, the contrast variation approach can reveal shapes of distinct components within a macromolecular complex.The goal of this thesis is to explore unchartered territories of regulatory protein-DNA interactions by studying such complexes by SAS, with a specific focus on the flexibility of the complexes. In my study of the MexR-DNA complex, I try to elucidate the molecular mechanism by which the MexR repressor regulates the expression of the MexAB-OPrM efflux pump through DNA binding. This pump is one of the multidrug-resistant tools of the pathogen Pseudomonas Aeruginosa (P. Aer.). It can extrude antibacterial drugs from the bacteria enabling them to survive in hostile environments. In the second project, I strive to explore the MYC:MAX:DNA complex. This heterodimer assembly functions as a central hub in cellular growth control by regulating many biological functions, including proliferation, apoptosis, differentiation and transformation. Overexpression or deregulation of MYC is observed in up to 70% of human aggressive cancer forms, including prostate and breast cancers. By combining SAS with biophysical methods, the work presented in this thesis reveals novel information on the shape and dynamics of biomolecular assemblies critical to health and disease.This thesis comprises five chapters, each dealing with a different aspect of the work in those years. The first chapter introduces the reader to the motivations of this research, and it will give the reader a brief state of the art of the two projects. In the second chapter, I will give you all the theoretical instruments to understand better all the methods used in this thesis, I write first to provide an overview regarding the proteins and their capability to bind other macromolecules. I then will exploit the basics of the small-angle technique, focusing on the neutron contrast variation: the fundamental technique used throughout this thesis and the ab-initio modelling.In the third chapter, Methods, I will discuss the SAS measurements and the requirements for the experiments themselves, the procedure for the data reduction and the data processing and analysis to obtain the structural information.The fourth chapter is a summary of the results of the submitted papers and my contributions:Small-angle X-ray and neutron scattering of MexR and its complex with DNA supports a conformational selection binding modelResolving the DNA interaction of the MexR antibiotic resistance regulatory proteinUpgraded D22 SEC-SANS set-up dedicated to the biology communitySAS studies on the regulation of MYC303:MAX:DNA and MAX:MAX:DNA binding in cancer.
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| 6. |
- Fernandes, Aristi Potamitou, et al.
(författare)
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A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:26, s. 24544-24552
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Tidskriftsartikel (refereegranskat)abstract
- Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3′,5′-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.
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| 7. |
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| 8. |
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| 9. |
- Hennig, Janosch, 1977-, et al.
(författare)
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MTMDAT : Automated analysis and visualization of mass spectrometry data for tertiary and quaternary structure probing of proteins
- 2008
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Ingår i: Bioinformatics. - : Oxford University Press (OUP). - 1367-4803 .- 1367-4811. ; 24:10, s. 1310-1312
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Tidskriftsartikel (refereegranskat)abstract
- In structural biology and -genomics, nuclear magnetic resonance (NMR) spectroscopy and crystallography are the methods of choice, but sample requirements can be hard to fulfil. Valuable structural information can also be obtained by using a combination of limited proteolysis and mass spectrometry, providing not only knowledge of how to improve sample conditions for crystallization trials or NMR spectrosopy by gaining insight into subdomain identities but also probing tertiary and quaternary structure, folding and stability, ligand binding, protein interactions and the location of post-translational modifications. For high-throughput studies and larger proteins, however, this experimentally fast and easy approach produces considerable amounts of data, which until now has made the evaluation exceedingly laborious if at all manually possible. MTMDAT, equipped with a browser-like graphical user interface, accelerates this evaluation manifold by automated peak picking, assignment, data processing and visualization. © 2008 The Author(s).
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| 10. |
- Johansson-Åkhe, Isak, 1993-
(författare)
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Development and Application of Computational Models for Peptide-Protein Complexes
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein-protein interactions between a protein and a smaller protein fragment or a disordered segment of a protein are called peptide-protein interactions. Such interactions are commonplace in nature and vital for normal cell function in humans. For example, the onco-protein Myc con- tains a large disordered region with several segments involved in peptide-protein interactions as part of transcription regulation, and it is mis-regulated in the vast majority of all human can- cers. As such, understanding the structural details of peptide-protein interactions on an atomic level is a necessary endeavor for understanding disease pathways as well as facilitating targeted drug-design. While experimental methods for structure determination such as X-ray crystallography and NMR can determine the structure of many peptide-protein complexes, these methods are time- consuming and costly. Additionally, the disordered nature of peptides and a sometimes lower binding affinity than for protein-protein binding can lead to transient or weak (but still highly specific) interactions impossible to fully capture with experimental methods. This leads to the need for computational methods as support and complement. Such methods have classically used statistical potentials or simple template search approaches, but as the number of deposited structures in the protein databank (PDB) grows so does the potential for supervised machine learning. The papers in this thesis present the contributions of the author to the field of peptide-protein in- teraction complex prediction, mainly through use of machine learning models. The first papers apply a Random Forest classifier to detect similarities between binding interfaces deposited in the PDB and a peptide-protein pair being investigated to find the optimal templates for struc- ture prediction. In excess of producing predictions with good self-evaluation of performance, the development of the method also confirmed theories on the similarity of protein-protein, domain-domain, and peptide-protein interfaces. Two more method for peptide-protein docking are presented in later papers. One utilizes graph convolution neural networks to improve model selection from rigid-body-docking methods by including MSA profile information as a feature, which also lead to the discovery that while profile information such as position conservation does improve predictive performance, something also seen in the first papers, the most impor- tant features are the ones describing the structural details of the complex and the bonds between residues. The other uses a graph neural network as an additional scoring term to improve upon the already state-of-the-art performing local refinement method FlexPepDock, and is capable of refining even models generated by AlphaFold-multimer. Finally, two manuscripts focus on the application of computational approaches for research into the interactions of human cMyc with TBP and PPP1R10, respectively. In the first of these pa- pers, the template-based peptide-protein complex prediction methods developed in the earlier papers of the thesis are employed together with prior knowledge of the interaction to model the complex to a high degree of certainty not achievable by NMR alone. In the second of these papers, experimental data is used as a basis for computational modeling of the complex, and the modeled complex could act as a basis for further experiments characterizing the interaction.
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