| 1. |
- Bernado, Pau, et al.
(författare)
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Structure and Dynamics of Ribosomal Protein L12: An Ensemble Model Based on SAXS and NMR Relaxation
- 2010
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Ingår i: Biophysical Journal. - : Elsevier BV. - 1542-0086 .- 0006-3495. ; 98:10, s. 2374-2382
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Tidskriftsartikel (refereegranskat)abstract
- Ribosomal protein L12 is a two-domain protein that forms dimers mediated by its N-terminal domains. A 20-residue linker separates the N- and C-terminal domains. This linker results in a three-lobe topology with significant flexibility, known to be critical for efficient translation. Here we present an ensemble model of spatial distributions and correlation times for the domain reorientations of L12 that reconciles experimental data from small-angle x-ray scattering and nuclear magnetic resonance. We generated an ensemble of L12 conformations in which the structure of each domain is fixed but the domain orientations are variable. The ensemble reproduces the small-angle x-ray scattering data and the optimized correlation times of its reorientational eigenmodes fit the N-15 relaxation data. The ensemble model reveals intrinsic conformational properties of L12 that help explain its function on the ribosome. The two C-terminal domains sample a large volume and extend further away from the ribosome anchor than expected for a random-chain linker, indicating that the flexible linker has residual order. Furthermore, the distances between each C-terminal domain and the anchor are anticorrelated, indicating that one of them is more retracted on average. We speculate that these properties promote the function of L12 to recruit translation factors and control their activity on the ribosome.
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| 2. |
- Blobel, Jascha, et al.
(författare)
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Protein loop compaction and the origin of the effect of arginine and glutamic acid mixtures on solubility, stability and transient oligomerization of proteins
- 2011
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Ingår i: European Biophysics Journal. - : Springer Science and Business Media LLC. - 0175-7571 .- 1432-1017. ; 40:12, s. 1327-1338
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Tidskriftsartikel (refereegranskat)abstract
- Addition of a 50 mM mixture of l-arginine and l-glutamic acid (RE) is extensively used to improve protein solubility and stability, although the origin of the effect is not well understood. We present Small Angle X-ray Scattering (SAXS) and Nuclear Magnetic Resonance (NMR) results showing that RE induces protein compaction by collapsing flexible loops on the protein core. This is suggested to be a general mechanism preventing aggregation and improving resistance to proteases and to originate from the polyelectrolyte nature of RE. Molecular polyelectrolyte mixtures are expected to display long range correlation effects according to dressed interaction site theory. We hypothesize that perturbation of the RE solution by dissolved proteins is proportional to the volume occupied by the protein. As a consequence, loop collapse, minimizing the effective protein volume, is favored in the presence of RE.
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| 3. |
- Grela, Przemysłw, et al.
(författare)
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Structural characterization of the ribosomal P1A-P2B protein dimer by small-angle X-ray scattering and NMR spectroscopy
- 2007
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 46:7, s. 1988-1998
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Tidskriftsartikel (refereegranskat)abstract
- The five ribosomal P-proteins, denoted P0-(P1-P2)(2), constitute the stalk structure of the large subunit of eukaryotic ribosomes. In the yeast Saccharomyces cerevisiae, the group of P1 and P2 proteins is differentiated into subgroups that form two separate P1A-P2B and P1B-P2A heterodimers on the stalk. So far, structural studies on the P-proteins have not yielded any satisfactory information using either X-ray crystallography or NMR spectroscopy, and the structures of the ribosomal stalk and its individual constituents remain obscure. Here we outline a first, coarse-grained view of the P1A-P2B solution structure obtained by a combination of small-angle X-ray scattering and heteronuclear NMR spectroscopy. The complex has an elongated shape with a length of 10 nm and a cross section of similar to 2.5 nm. N-15 NMR relaxation measurements establish that roughly 30% of the residues are present in highly flexible segments, which belong primarily to the linker region and the C-terminal part of the polypeptide chain. Secondary structure predictions and NMR chemical shift analysis, together with previous results from CD spectroscopy, indicate that the structured regions involve alpha-helices. NMR relaxation data further suggest that several helices are arranged in a nearly parallel or antiparallel topology. These results provide the first structural comparison between eukaryotic P1 and P2 proteins and the prokaryotic L12 counterpart, revealing considerable differences in their overall shapes, despite similar functional roles and similar oligomeric arrangements. These results present for the first time a view of the structure of the eukaryotic stalk constituents, which is the only domain of the eukaryotic ribosome that has escaped successful structural characterization.
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| 4. |
- Lazar, Tamas, et al.
(författare)
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PED in 2021 : A major update of the protein ensemble database for intrinsically disordered proteins
- 2021
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 49:D1, s. 404-411
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Tidskriftsartikel (refereegranskat)abstract
- The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.
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| 5. |
- Nasedkin, Alexandr
(författare)
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Tracing Molecular Conformations by X-ray Solution Scattering
- 2014
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- In the present work the refinement of X-ray data have been used to solve different scientific problems, namely drug localization in the liposome wall, reaction kinetics of the small molecule upon excitation and resolving an ensemble of protein structures at different temperatures. The common approach included collection of X-ray scattering patterns, modelling of the system of interest in atomic level and fitting computed results to experimental data. Employed fitting algorithms varied depends on the application ranging from inverse matrix calculations to the genetic algorithm for complex tasks. Observed results broaden our understanding of investigated systems on molecular level and also lead to development faster, more effective ways to sample atomic structures by X-ray diffusion scattering.
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| 6. |
- Orellana, Laura, et al.
(författare)
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Oncogenic mutations at the EGFR ectodomain structurally converge to remove a steric hindrance on a kinase-coupled cryptic epitope
- 2019
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : NATL ACAD SCIENCES. - 0027-8424 .- 1091-6490. ; 116:20, s. 10009-10018
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Tidskriftsartikel (refereegranskat)abstract
- Epidermal growth factor receptor (EGFR) signaling is initiated by a large ligand-favored conformational change of the extracellular domain (ECD) from a closed, self-inhibited tethered monomer, to an open untethered state, which exposes a loop required for strong dimerization and activation. In glioblastomas (GBMs), structurally heterogeneous missense and deletion mutations concentrate at the ECD for unclear reasons. We explore the conformational impact of GBM missense mutations, combining elastic network models (ENMs) with multiple molecular dynamics (MD) trajectories. Our simulations reveal that the main missense class, located at the I-II interface away from the self-inhibitory tether, can unexpectedly favor spontaneous untethering to a compact intermediate state, here validated by small-angle X-ray scattering (SAXS). Significantly, such intermediate is characterized by the rotation of a large ECD fragment (N-TR1), deleted in the most common GBM mutation, EGFRvIII, and that makes accessible a cryptic epitope characteristic of cancer cells. This observation suggested potential structural equivalence of missense and deletion ECD changes in GBMs. Corroborating this hypothesis, our FACS, in vitro, and in vivo data demonstrate that entirely different ECD variants all converge to remove N-TR1 steric hindrance from the 806-epitope, which we show is allosterically coupled to an intermediate kinase and hallmarks increased oncogenicity. Finally, the detected extraintracellular coupling allows for synergistic cotargeting of the intermediate with mAb806 and inhibitors, which is proved herein.
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| 7. |
- Salomonsson, Johannes, 1991-
(författare)
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Structural, Dynamic and Functional Studies of USP14 : A Proteasome-Associated Deubiquitinating Enzyme Regulating Protein Degradation
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Ubiquitin-Specific Protease 14 (USP14) is a proteasome-associated deubiquitinating enzyme (DUB) involved in regulation of protein degradation and recycling of ubiquitin. USP14 consists of a Ubl domain connected to a USP domain via a flexible linker. The Ubl domain is believed to be responsible for initial association to Rpn1 on the 19S proteasome while the USP domain binds ubiquitin and cleaves the isopeptide bond in ubiquitin chains. Due to its prevalence in various cancers, USP14 has been identified as a promising target for drug development and has been shown to decrease tumor growth and dissemination in zebrafish when inhibited by small molecules.The structure of the USP14 USP domain has been characterized by X-ray crystallography in its free state as well as when bound to ubiquitin or catalytic inhibitors. Additionally, structures of full-length USP14 in complex with the proteasome and ubiquitin have been solved by cryo- EM. However, by these methods, only stable structural elements are visible, limiting the understanding of the dynamic properties of USP14.In this thesis, I have used a wide range of biophysical analysis methods together with nuclear magnetic resonance (NMR) to study the structural, functional, and dynamic properties of USP14. In collaboration with pharmacologists, I have applied biophysical analysis and modelling to the analysis of a library of USP14 binders to elucidate binding affinities and specificities. By combining NMR with small-angle X-ray scattering, I have described the dynamic interaction between the USP14 Ubl domain and its catalytic USP domain and its predisposition toward proteasome binding. Deuteration of USP14 and full NMR analysis of the dynamic properties of the USP domain in the presence and absence of the Ubl domain provided extended understanding of how dynamic networks could be involved in regulating the catalytic activities of USP14. Finally, NMR analysis of USP14 non-Ub-binding mutants jointly with functional analysis has extended our understanding on how USP14 regulates the activities of the proteasome. Taken together, the studies presented here contribute to the investigation of novel therapeutic pathways designed to improve cancer treatment and patient care.
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