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| 2. |
- Fedorov, R V, et al.
(författare)
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Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins
- 2001
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D57:7, s. 968-976
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Tidskriftsartikel (refereegranskat)abstract
- The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 Å resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven -strands connected by long loops. TL5 binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd2+ ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TL5 sequence reveals homology to the so-called general stress protein CTC. The hydrophobic cores which stabilize both TL5 domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5.
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| 3. |
- Kristensen, Ole, et al.
(författare)
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Crystallization of a stringent response factor from Aquifex aeolicus.
- 2002
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; 58:Pt 7, s. 1198-1200
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Tidskriftsartikel (refereegranskat)abstract
- The crystallization of a key enzyme from Aquifex aeolicus with suggested bifunctional activity, acting as an exopolyphosphatase and a guanosine pentaphosphate phosphohydrolase, is reported. Native data were collected to below 2 A resolution from an orthorhombic crystal with unit-cell parameters a = 50.8, b = 70.3, c = 90.9 A. Methionine residues were introduced by mutation and deliberate oxidation of the protein allowed us to produce additional crystal forms with reproducible diffraction ability and increased phasing potential. This is the first report on the crystallization of a member of the Ppx/GppA phosphatase family.
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| 4. |
- Kristensen, Ole, et al.
(författare)
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Expression, refolding and crystallization of Aquifex aeolicus elongation factor P.
- 2002
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D58:Pt 6 Nr 2, s. 1039-1041
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Tidskriftsartikel (refereegranskat)abstract
- Elongation factor P is a universally conserved protein stimulating peptidyltransferase activity during protein synthesis. The factor is sensitive to classical inhibitors of the ribosomal peptidyltransferase activity and is possibly involved in alignment of the substrate tRNAs in the catalytic centre of 70S ribosomes. Elongation factor P from the thermophilic Aquifex aeolicus was overexpressed as a soluble protein in Escherichia coli and crystallized. A fast generally applicable refolding protocol was developed to improve crystal quality and circumvent strong binding of oligonucleotides to the protein. Diffraction data collected to 2.7 A resolution present twinning.
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| 5. |
- Kristensen, O, et al.
(författare)
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Is tRNA binding or tRNA mimicry mandatory for translation factors?
- 2002
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Ingår i: Current Protein and Peptide Science. - 1875-5550. ; 3:1, s. 133-141
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Forskningsöversikt (refereegranskat)abstract
- tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the: A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that, translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.
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| 6. |
- Kristensen, O, et al.
(författare)
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Structural characterization of the stringent response related exopolyphosphatase/guanosine pentaphosphate phosphohydrolase protein family
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:28, s. 8894-8900
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Tidskriftsartikel (refereegranskat)abstract
- Exopolyphosphatase/guanosine pentaphosphate phosphohydrolase (PPX/GPPA) enzymes play central roles in the bacterial stringent response induced by starvation. The high-resolution crystal structure of the putative Aquifex aeolicus PPX/GPPA phosphatase from the actin-like ATPase domain superfamily has been determined, providing the first insights to features of the common catalytic core of the PPX/GPPA family. The protein has a two-domain structure with an active site located in the interdomain cleft. Two crystal forms were investigated (type I and 11) at resolutions of 1.53 and 2.15 Angstrom, respectively. This revealed a structural flexibility that has previously been described as a "butterfly-like" cleft opening around the active site in other actin-like superfamily proteins. A calcium ion is observed at the center of this region in type I crystals, substantiating that PPX/GPPA enzymes use metal ions for catalysis. Structural analysis suggests that nucleotides bind at a similar position to that seen in other members of the superfamily.
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| 7. |
- Laurberg, Martin
(författare)
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Dynamics in Protein Synthesis, Structural Studies of Translation Factors
- 2002
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Protein biosynthesis is performed on ribosomes. The ribosome is a complex of ribosomal RNA and proteins forming two subunits. A messenger RNA strand is decoded on the small subunit by charged aminoacyl transfer RNA (aa-tRNA) molecules and each tRNA molecule delivers its amino acid to the growing polypeptide chain on the large subunit. RNA performs the central reactions and ribosomal proteins and factors assist in the process. The elongation factors EF-Tu and EF-G are GTPases alternately binding to the ribosomal factor-binding site. EF-Tu inserts an aa-tRNA molecule in the ribosomal tRNA acceptor site (A-site) and EF-G catalyzes the synchronized translocation of tRNAs and the messenger between ribosomal sites. The crystal structure of EF-G carrying the His573Ala mutation has been determined and it revealed the structure of domain III of EF-G. This domain extends the tRNA L-shape mimic recurrently observed among translation factors to include the tRNA acceptor stem. An observed rotation of domains III, IV and V versus domains I and II brings domains III and I in close contact. Mutations conferring strong resistance towards the antibiotic fusidic acid (FA) have been identified to flank this domains interface and a possible FA binding site has been identified. Impairment by FA after GTP hydrolysis and structural rearrangements of EF-G have been proposed to prevent the factor from dissociating after translocation and thereby block protein synthesis. The crystal structure of a FA hypersensitive mutant with an increased affinity for GTP supports this view. During acute amino acid starvation the ribosome associated stringent factor RelA catalyses the formation of pppGpp from ATP and GTP as a response to an elevated frequency of uncharged tRNAs in the ribosomal A-site. The stringent response factor, GPP, hydrolyzes pppGpp to the active ppGpp affecting levels of transcription, protein synthesis and protein degradation. GPP has successfully been crystallized and structure determination is currently under way.The elongation factor P catalyzing formation of the first peptide bond of the nascent peptide chain on the ribosome has been crystallized and structure determination is in progress.
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| 8. |
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| 9. |
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| 10. |
- Willows, R D, et al.
(författare)
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Crystallization and preliminary X-ray analysis of the Rhodobacter capsulatus magnesium chelatase BchI subunit
- 1999
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D55, s. 689-690
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Tidskriftsartikel (refereegranskat)abstract
- The Rhodobacter capsulatus BchI protein is one of three subunits of Mg chelatase, the enzyme which catalyzes the first committed step of chlorophyll and bacteriochlorophyll biosynthesis. The BchI protein was produced with an inducible T7 RNA polymerase expression system in Escherichia coli. The protein was purified from the soluble cell-extract fraction and crystallized from polyethylene glycol solution. The crystals diffract to a minimum Bragg spacing of 2.1 Å. The space group is P63 with unit-cell dimensions a = b = 90.6, c = 84.1 Å.
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