| 1. |
- Hederos, Sofia, et al.
(författare)
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A new enzyme by rational design - the incorporation of a single His residue enables efficient thioester hydrolysis by human glutathione transferase A1-1
- 2004
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Ingår i: Proc. Nat. Acad. Sci.. ; 101, s. 13163-13167
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Tidskriftsartikel (refereegranskat)abstract
- A strategy for rational enzyme design is reported and illustrated by the engineering of a protein catalyst for thiol-ester hydrolysis. Five mutants of human glutathione (GSH; gamma-Glu-Cys-Gly) transferase A1-1 were designed in the search for a catalyst and to provide a set of proteins from which the reaction mechanism could be elucidated. The single mutant A216H catalyzed the hydrolysis of the S-benzoyl ester of GSH under turnover conditions with a k(cat)/K(M) of 156 M(-1) x min(-1), and a catalytic proficiency of >10(7) M(-1) when compared with the first-order rate constant of the uncatalyzed reaction. The wild-type enzyme did not hydrolyze the substrate, and thus, the introduction of a single histidine residue transformed the wild-type enzyme into a turnover system for thiol-ester hydrolysis. By kinetic analysis of single, double, and triple mutants, as well as from studies of reaction products, it was established that the enzyme A216H catalyzes the hydrolysis of the thiol-ester substrate by a mechanism that includes an acyl intermediate at the side chain of Y9. Kinetic measurements and the crystal structure of the A216H GSH complex provided compelling evidence that H216 acts as a general-base catalyst. The introduction of a single His residue into human GSH transferase A1-1 created an unprecedented enzymatic function, suggesting a strategy that may be of broad applicability in the design of new enzymes. The protein catalyst has the hallmarks of a native enzyme and is expected to catalyze various hydrolytic, as well as transesterification, reactions.
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| 2. |
- Dalhus, Bjørn, et al.
(författare)
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Structural basis for thermophilic protein stability : structures of thermophilic and mesophilic malate dehydrogenases.
- 2002
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Ingår i: J Mol Biol. - 0022-2836. ; 318:3, s. 707-21
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Tidskriftsartikel (refereegranskat)abstract
- The three-dimensional structure of four malate dehydrogenases (MDH) from thermophilic and mesophilic phototropic bacteria have been determined by X-ray crystallography and the corresponding structures compared. In contrast to the dimeric quaternary structure of most MDHs, these MDHs are tetramers and are structurally related to tetrameric malate dehydrogenases from Archaea and to lactate dehydrogenases. The tetramers are dimers of dimers, where the structures of each subunit and the dimers are similar to the dimeric malate dehydrogenases. The difference in optimal growth temperature of the corresponding organisms is relatively small, ranging from 32 to 55 degrees C. Nevertheless, on the basis of the four crystal structures, a number of factors that are likely to contribute to the relative thermostability in the present series have been identified. It appears from the results obtained, that the difference in thermostability between MDH from the mesophilic Chlorobium vibrioforme on one hand and from the moderate thermophile Chlorobium tepidum on the other hand is mainly due to the presence of polar residues that form additional hydrogen bonds within each subunit. Furthermore, for the even more thermostable Chloroflexus aurantiacus MDH, the use of charged residues to form additional ionic interactions across the dimer-dimer interface is favored. This enzyme has a favorable intercalation of His-Trp as well as additional aromatic contacts at the monomer-monomer interface in each dimer. A structural alignment of tetrameric and dimeric prokaryotic MDHs reveal that structural elements that differ among dimeric and tetrameric MDHs are located in a few loop regions. (c) 2002 Elsevier Science Ltd.
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| 3. |
- Hallberg, Eric, et al.
(författare)
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Sensilla and proprioceptors
- 2003
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Ingår i: Lepidoptera, Moths and Butterflies : Volume 2: Morphology, Physiology, and Development - Volume 2: Morphology, Physiology, and Development. - : DE GRUYTER. - 9783110893724 - 3110162105 - 9783110162103 ; , s. 267-288
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Bokkapitel (refereegranskat)
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| 4. |
- Lundström, Patrik, 1971-, et al.
(författare)
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Quantitative analysis of conformational exchange contributions to H-1-N-15 multiple-quantum relaxation using field-dependent measurements. Time scale and structural characterization of exchange in a calmodulin C-terminal domain mutant
- 2004
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Ingår i: Journal of the American Chemical Society. - : American Chemical Society (ACS). - 0002-7863 .- 1520-5126. ; 126:3, s. 928-935
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Tidskriftsartikel (refereegranskat)abstract
- Multiple-quantum spin relaxation is a sensitive probe for correlated conformational exchange dynamics on microsecond to millisecond time scales in biomolecules. We measured differential H-1-N-15 multiple-quantum relaxation rates for the backbone amide groups of the E140Q mutant of the C-terminal domain of calmodulin at three static magnetic field strengths. The differential multiple-quantum relaxation rates range between -88.7 and 92.7 s(-1), and the mean and standard deviation are 7.0 24 s(-1), at a static magnetic field strength of 14.1 T. Together with values of the H-1 and N-15 chemical shift anisotropies (CSA) determined separately, the field-dependent data enable separation of the different contributions from dipolar-dipolar, CSA-CSA, and conformational exchange cross-correlated relaxation mechanisms to the differential multiple-quantum relaxation rates. The procedure yields precise quantitative information on the dominant conformational exchange contributions observed in this protein. The field-dependent differences between double- and zero-quantum relaxation rates directly benchmark the rates of conformational exchange, showing that these are fast on the chemical shift time scale for the large majority of residues in the protein. Further analysis of the differential H-1-N-15 multiple-quantum relaxation rates using previously determined exchange rate constants and populations, obtained from N-15 off-resonance rotating-frame relaxation data, enables extraction of the product of the chemical shift differences between the resonance frequencies of the H-1 and N-15 spins in the exchanging conformations, deltasigma(H)deltasigma(N). Thus, information on the H-1 chemical shift differences is obtained, while circumventing complications associated with direct measurements of conformational exchange effects on H-1 single-quantum coherences in nondeuterated proteins. The method significantly increases the information content available for structural interpretation of the conformational exchange process, partly because deltasigma(H)deltasigma(N) is a signed quantity, and partly because two chemical shifts are probed simultaneously. The present results support the hypothesis that the exchange in the calcium-loaded state of the E140Q mutant involves conformations similar to those of the wild-type apo (closed) and calcium-loaded (open) states.
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| 5. |
- Wolf-Watz, Magnus, et al.
(författare)
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Chloride binding by the AML1/Runx1 transcription factor studied by NMR
- 2001
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Ingår i: FEBS Letters. - 0014-5793. ; 488:1-2, s. 81-4
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Tidskriftsartikel (refereegranskat)abstract
- It is known that the DNA binding Runt domain of the AML1/Runx1 transcription factor coordinates Cl(-) ions. In this paper we have determined Cl(-) binding affinities of AML1 by (35)Cl nuclear magnetic resonance (NMR) linewidth analysis. The Runt domain binds Cl(-) with a dissociation constant (K(d,Cl)) of 34 mM. If CBFbeta is added to form a 1:1 complex, the K(d,Cl) value increases to 56 mM. Homology modeling suggests that a high occupancy Cl(-) binding site overlaps with the DNA binding surface. NMR data show that DNA displaces this Cl(-) ion. Possible biological roles of Cl(-) binding are discussed based on these findings.
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| 6. |
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| 7. |
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| 8. |
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| 9. |
- Pratt, J. R., et al.
(författare)
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Effects of methylphosphonate, a phosphate analogue, on the expression and degradation of the high-affinity phosphate transporter Pho84, in Saccharomyces cerevisiae
- 2004
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 43:45, s. 14444-53
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Tidskriftsartikel (refereegranskat)abstract
- In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Deltapho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by (31)P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.
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| 10. |
- Andersson, B, et al.
(författare)
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Crystallization and X-ray diffraction data analysis of leukotriene A4 hydrolase from Saccharomyces cerevisiae
- 2003
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D59:Pt 6, s. 1093-1095
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Tidskriftsartikel (refereegranskat)abstract
- The Saccharomyces cerevisiae leukotriene A4 (LTA4) hydrolase (scLTA4 hydrolase) has been crystallized in order to study the two activities of LTA4 hydrolase in an evolutionary perspective. Single well diffracting crystals are obtained after switching from the hanging-drop method to liquid-liquid diffusion in capillaries using PEG 8000 as precipitant. These crystals belong to space group P212121, with unit-cell parameters a = 70.8, b = 98.1, c = 99.2 Å. Intensity data to 2.3 Å resolution were collected from a native scLTA4 hydrolase crystal using synchrotron radiation. A molecular-replacement solution was obtained using the human LTA4 hydrolase structure and the program BEAST.
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