| 1. |
- Costeira-Paulo, Joana, et al.
(författare)
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Lipids Shape the Electron Acceptor-Binding Site of the Peripheral Membrane Protein Dihydroorotate Dehydrogenase
- 2018
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 25:3, s. 309-317
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Tidskriftsartikel (refereegranskat)abstract
- The interactions between proteins and biological membranes are important for drug development, but remain notoriously refractory to structural investigation. We combine non-denaturing mass spectrometry (MS) with molecular dynamics (MD) simulations to unravel the connections among co-factor, lipid, and inhibitor binding in the peripheral membrane protein dihydroorotate dehydrogenase (DHODH), a key anticancer target. Interrogation of intact DHODH complexes by MS reveals that phospholipids bind via their charged head groups at a limited number of sites, while binding of the inhibitor brequinar involves simultaneous association with detergent molecules. MD simulations show that lipids support flexible segments in the membrane-binding domain and position the inhibitor and electron acceptor-binding site away from the membrane surface, similar to the electron acceptor-binding site in respiratory chain complex I. By complementing MS with MD simulations, we demonstrate how a peripheral membrane protein uses lipids to modulate its structure in a similar manner as integral membrane proteins.
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| 2. |
- Kaldmäe, Margit, et al.
(författare)
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A “spindle and thread” mechanism unblocks p53 translation by modulating N-terminal disorder
- 2022
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Ingår i: Structure. - : Elsevier BV. - 0969-2126 .- 1878-4186. ; 30:5, s. 733-742, e1-e7
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Tidskriftsartikel (refereegranskat)abstract
- Disordered proteins pose a major challenge to structural biology. A prominent example is the tumor suppressor p53, whose low expression levels and poor conformational stability hamper the development of cancer therapeutics. All these characteristics make it a prime example of “life on the edge of solubility.” Here, we investigate whether these features can be modulated by fusing the protein to a highly soluble spider silk domain (NT∗). The chimeric protein displays highly efficient translation and is fully active in human cancer cells. Biophysical characterization reveals a compact conformation, with the disordered transactivation domain of p53 wrapped around the NT∗ domain. We conclude that interactions with NT∗ help to unblock translation of the proline-rich disordered region of p53. Expression of partially disordered cancer targets is similarly enhanced by NT∗. In summary, we demonstrate that inducing co-translational folding via a molecular “spindle and thread” mechanism unblocks protein translation in vitro.
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| 3. |
- Kronqvist, Nina, et al.
(författare)
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Efficient protein production inspired by how spiders make silk
- 2017
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.
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| 4. |
- Kronqvist, Nina, et al.
(författare)
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Sequential pH-driven dimerization and stabilization of the N-terminal domain enables rapid spider silk formation
- 2014
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 5:1, s. 3254-
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Tidskriftsartikel (refereegranskat)abstract
- The mechanisms controlling the conversion of spider silk proteins into insoluble fibres, which happens in a fraction of a second and in a defined region of the silk glands, are still unresolved. The N-terminal domain changes conformation and forms a homodimer when pH is lowered from 7 to 6; however, the molecular details still remain to be determined. Here we investigate site-directed mutants of the N-terminal domain from Euprosthenops australis major ampullate spidroin 1 and find that the charged residues D40, R60 and K65 mediate intersubunit electrostatic interactions. Protonation of E79 and E119 is required for structural conversions of the subunits into a dimer conformation, and subsequent protonation of E84 around pH 5.7 leads to the formation of a fully stable dimer. These residues are highly conserved, indicating that the now proposed three-step mechanism prevents premature aggregation of spidroins and enables fast formation of spider silk fibres in general.
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| 5. |
- Sarr, Medoune, et al.
(författare)
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A spidroin-derived solubility tag enables controlled aggregation of a designed amyloid protein
- 2018
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Ingår i: The FEBS Journal. - : WILEY. - 1742-464X .- 1742-4658. ; 285:10, s. 1873-1885
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Tidskriftsartikel (refereegranskat)abstract
- Amyloidogenesis is associated with more than 30 diseases, but the molecular mechanisms involved in cell toxicity and fibril formation remain largely unknown. The inherent tendency of amyloid-forming proteins to aggregate renders expression, purification, and experimental studies challenging. NT* is a solubility tag derived from a spider silk protein that was recently introduced for the production of several aggregation-prone peptides and proteins at high yields. Herein, we investigate whether fusion to NT* can prevent amyloid fibril formation and enable controlled aggregation for experimental studies. As an example of an amyloidogenic protein, we chose the de novo-designed polypeptide 17. The fusion protein NT*-17 was recombinantly expressed in Escherichia coli to produce high amounts of soluble and mostly monomeric protein. Structural analysis showed that 17 is kept in a largely unstructured conformation in fusion with NT*. After proteolytic release, 17 adopts a -sheet conformation in a pH- and salt-dependent manner and assembles into amyloid-like fibrils. The ability of NT* to prevent premature aggregation and to enable structural studies of prefibrillar states may facilitate investigation of proteins involved in amyloid diseases.
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| 6. |
- Sarr, Médoune
(författare)
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A star is born : development of NT* as a new biotechnological tool based on the mechanism of pH dependent dimerization of the spidroin N-terminal domain
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Proteins are widely used in research and in the pharmaceutical industry but the production of recombinant protein can be tedious, costly and time-consuming due to unwanted aggregation. Strategies to circumvent aggregation, e.g. the use of solubility tags, must be evaluated experimentally and successful results are not always obtained. Therefore, there is a need to develop novel solubility tags for problematic proteins. In this thesis, we propose that spiders’ own solubility tag – the N-terminal domain (NT) of the spider silk proteins (spidroins) –– can be harnessed to produce very aggregation-prone proteins. NT is a pH-sensitive relay and its conformational switch from monomer to dimer determine the assembly state of the spider silk proteins. However, the molecular mechanisms of NT dimerization are unclear. In this thesis we determined which residues regulate the dimerization of NT from major ampullate spidroin and investigated whether this mechanism is conserved between distantly related NTs. Our results showed that NT dimerization requires an initial electrostatic interaction mediated by aspartate 40 with lysine 65, and that upon lowering the pH, subsequent protonation of glutamates 79, 84 and 119 result in conformational changes and stabilization. Moreover, we provided evidence that charge attraction and multistep protonation is conserved between widely different NTs but is mediated by different sets of residues. On the basis of these findings, we designed the soluble and thermally stable NT* by disrupting the charge attraction between aspartate 40 and lysine 65. NT* was more efficient to promote solubility of aggregation-prone proteins and peptides than commonly used solubility tags. We hypothesized further that NT* would be a suitable tag to control the solubility of amyloidogenic proteins and, thereby prevent precocious aggregation. Our results support that NT* prevents aggregation of amyloidogenic proteins, allowing e.g. structural studies and identification of new anti-amyloid strategies. This thesis presents an entirely novel approach to produce aggregation-prone recombinant proteins using the biotechnological tool NT* which was conceived on the basis of how spiders store their spidroins at high concentration without precocious aggregation.
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