| 1. |
- Bergkvist, Anders, et al.
(författare)
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Surface interactions in the complex between cytochrome f and the E43Q/D44N and E59K/E60Q plastocyanin double mutants as determined by (1)H-NMR chemical shift analysis
- 2001
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Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 10:12, s. 2623-2626
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Tidskriftsartikel (refereegranskat)abstract
- A combination of site-directed mutagenesis and NMR chemical shift perturbation analysis of backbone and side-chain protons has been used to characterize the transient complex of the photosynthetic redox proteins plastocyanin and cytochrome f. To elucidate the importance of charged residues on complex formation, the complex of cytochrome f and E43Q/D44N or E59K/E60Q spinach plastocyanin double mutants was studied by full analysis of the (1)H chemical shifts by use of two-dimensional homonuclear NMR spectra. Both mutants show a significant overall decrease in chemical shift perturbations compared with wild-type plastocyanin, in agreement with a large decrease in binding affinity. Qualitatively, the E43Q/D44N mutant showed a similar interaction surface as wild-type plastocyanin. The interaction surface in the E59K/E60Q mutant was distinctly different from wild type. It is concluded that all four charged residues contribute to the affinity and that residues E59 and E60 have an additional role in fine tuning the orientation of the proteins in the complex.
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| 2. |
- Dunne, Aisling, et al.
(författare)
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Structural complementarity of Toll/interleukin-1 receptor domains in Toll-like receptors and the adaptors Mal and MyD88
- 2003
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Ingår i: Journal of Biological Chemistry. - : American Society for Biochemistry and Molecular Biology. - 0021-9258 .- 1083-351X. ; 278:42, s. 41443-41451
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Tidskriftsartikel (refereegranskat)abstract
- The Toll/interleukin 1 receptor (TIR) domain is a region found in the cytoplasmic tails of members of the Toll-like receptor/interleukin-1 receptor superfamily. The domain is essential for signaling and is also found in the adaptor proteins Mal (MyD88 adaptor-like) and MyD88, which function to couple activation of the receptor to downstream signaling components. Experimental structures of two Toll/interleukin 1 receptor domains reveal a alpha-beta-fold similar to that of the bacterial chemotaxis protein CheY, and other evidence suggests that the adaptors can make heterotypic interactions with both the receptors and themselves. Here we show that the purified TIR domains of Mal and MyD88 can form stable heterodimers and also that Mal homodimers and oligomers are dissociated in the presence of ATP. To identify structural features that may contribute to the formation of signaling complexes, we produced models of the TIR domains from human Toll-like receptor 4 (TLR4), Mal, and MyD88. We found that although the overall fold is conserved the electrostatic surface potentials are quite distinct. Docking studies of the models suggest that Mal and MyD88 bind to different regions in TLRs 2 and 4, a finding consistent with a cooperative role of the two adaptors in signaling. Mal and MyD88 are predicted to interact at a third non-overlapping site, suggesting that the receptor and adaptors may form heterotetrameric complexes. The theoretical model of the interactions is supported by experimental data from glutathione S-transferase pull-downs and co-immunoprecipitations. Neither theoretical nor experimental data suggest a direct role for the conserved proline in the BB-loop in the association of TLR4, Mal, and MyD88. Finally we show a sequence relationship between the Drosophila protein Tube and Mal that may indicate a functional equivalence of these two adaptors in the Drosophila and vertebrate Toll pathways.
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| 3. |
- Ejdebäck, Mikael, 1969-, et al.
(författare)
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Side-chain interactions in the plastocyanin-cytochrome f complex
- 2000
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 39:17, s. 5022-5027
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Tidskriftsartikel (refereegranskat)abstract
- Cytochrome f and plastocyanin are redox partners in the photosynthetic electron-transfer chain. Electron transfer from cytochrome f to plastocyanin occurs in a specific short-lived complex. To obtain detailed information about the binding interface in this transient complex, the effects of binding on the backbone and side-chain protons of plastocyanin have been analyzed by mapping NMR chemical-shift changes. Cytochrome f was added to plastocyanin up to 0.3 M equiv, and the plastocyanin proton chemical shifts were measured. Out of approximately 500 proton resonances, 86% could be observed with this method. Nineteen percent demonstrate significant chemical-shift changes and these protons are located in the hydrophobic patch (including the copper ligands) and the acidic patches of plastocyanin, demonstrating that both areas are part of the interface in the complex. This is consistent with the recently determined structure of the complex [Ubbink, M., Ejdebäck, M., Karlsson, B. G., and Bendall, D. S. (1998) Structure 6, 323-335]. The largest chemical-shift changes are found around His87 in the hydrophobic patch, which indicates tight contacts and possibly water exclusion from this part of the protein interface. These results support the idea that electron transfer occurs via His87 to the copper in plastocyanin and suggest that the hydrophobic patch determines the specificity of the binding. The chemical-shift changes in the acidic patches are significant but small, suggesting that the acidic groups are involved in electrostatic interactions but remain solvent exposed. The existence of small differences between the present data and those used for the structure may imply that the redox state of the metals in both proteins slightly affects the structure of the complex. The chemical-shift mapping is performed on unlabeled proteins, making it an efficient way to analyze effects of mutations on the structure of the complex.
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| 4. |
- Hardy, Matthew P., et al.
(författare)
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Characterization of the type I interferon locus and identification of novel genes
- 2004
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Ingår i: Genomics. - : Elsevier. - 0888-7543 .- 1089-8646. ; 84:2, s. 331-345
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Tidskriftsartikel (refereegranskat)abstract
- The human type I interferon (IFN) genes are clustered on human chromosome 9p21 and the mouse genes are located in the region of conserved synteny on mouse chromosome 4. We have identified two novel mouse Ifna genes (Ifna12, Ifna13) and Ifnl2 (IFN-like 2, a homologue of Limitin/IFN-like 1). Another type I IFN gene was designated Ifne1. Mouse Ifne1 was expressed in ovaries and uterus but not in tissues of hematopoietic origin. IFN-epsilon1 has general structural characteristics of a type I IFN. These studies represent the first detailed annotation of the mouse type I IFN locus, and the products of these novel genes may have important functions in reproduction and host defense.
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| 5. |
- Jansson, Hanna, 1975, et al.
(författare)
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The crystal structure of the spinach plastocyanin double mutant G8D/L12E gives insight into its low reactivity towards photosystem 1 and cytochrome f
- 2003
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0005-2728 .- 1879-2650. ; 1607:2-3, s. 203-210
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Tidskriftsartikel (refereegranskat)abstract
- Plastocyanin (Pc) is a copper-containing protein, which functions as an electron carrier between the cytochrome b6f and photosystem 1 (PS1) complexes in the photosynthetic electron transfer (ET) chain. The ET is mediated by His87 situated in the hydrophobic surface in the north region of Pc. Also situated in this region is Leu12, which mutated to other amino acids severely disturbs the ET from cytochrome f and to PS1, indicating the importance of the hydrophobic surface. The crystal structure of the Pc double mutant G8D/L12E has been determined to 2.0 Å resolution, with a crystallographic R-factor of 18.3% (Rfree=23.2%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. In particular, there is a small but significant change in the hydrophobic surface close to His87. Evidently, this leads to a mismatch in the reactive complex with the redox partners. For PS1 this results in a 20 times weaker binding and an eightfold slower ET as determined by kinetic measurements. The mutations that have been introduced do not affect the optical absorption spectrum. However, there is a small change in the EPR spectrum, which can be related to changes in the copper coordination geometry.
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| 6. |
- O'Neill, Luke A. J., et al.
(författare)
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Mal and MyD88 : adapter proteins involved in signal transduction by Toll-like receptors
- 2003
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Ingår i: Journal of Endotoxin Research. - : Maney Publishing. - 0968-0519 .- 1743-2839. ; 9:1, s. 55-59
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Tidskriftsartikel (refereegranskat)abstract
- Signal transduction processes activated by Toll-like receptors (TLRs) include the important transcription factor NF-kappaB and 2 MAP kinases, p38 and Jun N-terminal kinase. These signals ultimately give rise to increased expression of a multitude of pro-inflammatory proteins. Receptor-proximal proteins involved in signalling by all TLRs include the adapter MyD88, 3 IRAKs (IRAK-4, IRAK and IRAK-2), Tollip, Traf-6 and TAK-1. Differences between signals generated by TLRs are emerging, with both TLR4 and TLR2 signalling requiring an additional adapter termed MyD88-adapter-like (Mal; also known as TIRAP). MyD88 and Mal both have a homologous Toll/IL-1 receptor (TIR) domain although they differ in their N-termini, with MyD88 possessing a death domain. In addition, structural models reveal marked differences in surface charges which, when taken with surface charge differences between TLR2 and TLR4 TIR domains, may indicate that TLR4 but not TLR2 recruits Mal directly. Another difference is that Mal can become phosphorylated. Future studies on Mal will reveal specificities in signal transduction by different TLRs, which may ultimately provide molecular explanations for specificities in the innate immune response to infection.
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| 7. |
- Svensson, Maria, et al.
(författare)
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Functional prediction of a T-DNA tagged gene of Arabidopsis thalianaby in silico analysis
- 2004
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Ingår i: Journal of Molecular Modeling. - : Springer. - 1610-2940 .- 0948-5023. ; 10:2, s. 130-138
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Tidskriftsartikel (refereegranskat)abstract
- We have employed a gene-knockout approach using T-DNA tagging and in vivo gene fusion in Arabidopsis thaliana for identification and isolation of specific plant genes. Screening of about 3,000 T-DNA tagged lines resulted in identification of a mutant line (no. 197) exhibiting a significant delay in flowering. From this line a 600-bp plant DNA fragment downstream of the left T-DNA junction was cloned by inverse PCR. BLAST searching in the A. thaliana genomic database indicated a putative gene, frf (flowering regulating factor), with unknown function downstream of the T-DNA insert. Bioinformatic tools were used to predict possible protein structure and function. The protein structure predicted by fold recognition indicates that frf is a transcriptional regulator, a ligand-binding receptor responsive to steroids and hormones. Analyzing the predicted results and the phenotype of the T-DNA tagged plant we hypothesized that FRF might be involved in hormone response in A. thaliana. For verification of this hypothesis we exposed the plants of line no. 197 to gibberellic acid (GA3), a potential growth regulator in higher plants. This treatment resulted in an earlier onset of flowering, almost similar to that in wild type control plants.
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