| 51. |
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| 52. |
- Johansson, Patrik, et al.
(författare)
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Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
- 2006
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 15:10, s. 2300-2309
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Tidskriftsartikel (refereegranskat)abstract
- A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 angstrom. The Rv0130 monomer features a single hotdog fold composed of a highly curved beta-sheet on top of a long and a short alpha-helix. Two monomers in turn pack to form a double-hotdog-folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R-specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans-2-enoyl-coenzyme A moiety with a k(cat) of 1.1 x 10(2) sec(-1). The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site-directed mutagenesis. In contrast to many hotdog-folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate-binding channels of long-chain eukaryotic hydratase 2 enzymes.
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| 53. |
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| 54. |
- Kaldmae, Margit, et al.
(författare)
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A strategy for the identification of protein architectures directly from ion mobility mass spectrometry data reveals stabilizing subunit interactions in light harvesting complexes
- 2019
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Ingår i: Protein Science. - : WILEY. - 0961-8368 .- 1469-896X. ; 28:6, s. 1024-1030
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Tidskriftsartikel (refereegranskat)abstract
- Biotechnological applications of protein complexes require detailed information about their structure and composition, which can be challenging to obtain for proteins from natural sources. Prominent examples are the ring-shaped phycoerythrin (PE) and phycocyanin (PC) complexes isolated from the light-harvesting antennae of red algae and cyanobacteria. Despite their widespread use as fluorescent probes in biotechnology and medicine, the structures and interactions of their noncrystallizable central subunits are largely unknown. Here, we employ ion mobility mass spectrometry to reveal varying stabilities of the PC and PE complexes and identify their closest architectural homologues among all protein assemblies in the Protein Data Bank (PDB). Our results suggest that the central subunits of PC and PE complexes, although absent from the crystal structures, may be crucial for their stability, and thus of unexpected importance for their biotechnological applications.
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| 55. |
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| 56. |
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| 57. |
- Karlsson, Elin, et al.
(författare)
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High affinity between CREBBP/p300 and NCOA evolved in vertebrates
- 2020
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 29:7, s. 1687-1691
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Tidskriftsartikel (refereegranskat)abstract
- The interaction between the transcriptional coactivators CREBBP/p300 and NCOA is governed by two intrinsically disordered domains called NCBD and CID, respectively. The CID domain emerged within the NCOA protein in deuterostome animals (including vertebrates) after their split from the protostomes (molluscs, worms, and arthropods). However, it has not been clear at which point a high affinity interaction evolved within the deuterostome clade and whether all present-day deuterostome animals have a high affinity NCBD:CID interaction. We have here expressed and measured affinity for NCBD and CID domains from animal species representing different evolutionary branches of the deuterostome tree. While all vertebrate species have high-affinity NCBD:CID interactions we found that the interaction in the echinoderm purple sea urchin is of similar affinity as that of the proposed ancestral domains. Our findings demonstrate that the high-affinity NCBD:CID interaction likely evolved in the vertebrate branch and question whether the interaction between CREBBP/p300 and NCOA is essential in nonvertebrate deuterostomes. The data provide an example of evolution of transcriptional regulation through protein-domain based inventions.
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| 58. |
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| 59. |
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| 60. |
- Kelpšas, Vinardas, et al.
(författare)
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Enhancing protein perdeuteration by experimental evolution of Escherichia coli K-12 for rapid growth in deuterium-based media
- 2021
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 30:12, s. 2457-2473
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Tidskriftsartikel (refereegranskat)abstract
- Deuterium is a natural low abundance stable hydrogen isotope that in high concentrations negatively affects growth of cells. Here, we have studied growth of Escherichia coli MG1655, a wild-type laboratory strain of E. coli K-12, in deuterated glycerol minimal medium. The growth rate and final biomass in deuterated medium is substantially reduced compared to cells grown in ordinary medium. By using a multi-generation adaptive laboratory evolution-based approach, we have isolated strains that show increased fitness in deuterium-based growth media. Whole-genome sequencing identified the genomic changes in the obtained strains and show that there are multiple routes to genetic adaptation to growth in deuterium-based media. By screening a collection of single-gene knockouts of nonessential genes, no specific gene was found to be essential for growth in deuterated minimal medium. Deuteration of proteins is of importance for NMR spectroscopy, neutron protein crystallography, neutron reflectometry, and small angle neutron scattering. The laboratory evolved strains, with substantially improved growth rate, were adapted for recombinant protein production by T7 RNA polymerase overexpression systems and shown to be suitable for efficient production of perdeuterated soluble and membrane proteins for structural biology applications.
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