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- Knapp, S, et al.
(author)
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Thermal unfolding of small proteins with SH3 domain folding pattern
- 1998
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In: Proteins. - 0887-3585 .- 1097-0134. ; 31:3, s. 309-319
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Journal article (peer-reviewed)abstract
- The thermal unfolding of three SH3 domains of the Tec family of tyrosine kinases was studied by differential scanning calorimetry and CD spectroscopy, The unfolding transition of the three protein domains in the acidic pH region can be described as a reversible two-state process. For all three SH3 domains maximum stability was observed in the pH region 4.5 < pH < 7.0 where these domains unfold at temperatures of 353K (Btk), 342K (Itk), and 344K (Tec), At these temperatures an enthalpy change of 196 kJ/mol, 178 kJ/mol, and 169 kJ/mol was measured for Btk-, Itk-, and Tec-SH3 domains, respectively. The determined changes in heat capacity between the native and the denatured state are in an usual range expected for small proteins. Our analysis revealed that all SH3 domains studied are only weakly stabilized and have free energies of unfolding which do not exceed 12-16 kJ/mol but show quite high melting temperatures. Comparing unfolding free energies measured for eukaryotic SH3 domains with those of the topologically identical Sso7d protein from the hyperthermophile Sulfolobus solfataricus, the increased melting temperature of the thermostable protein is due to a broadening as well as a significant lifting of its stability curve. However, at their physiological temperatures, 310K for mesophilic SH3 domains and 350K for Sso7d, eukaryotic SH3 domains and Sso7d show very similar stabilities. (C) 1998 Wiley-Liss, Inc.
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| 3. |
- Oppermann, U C T, et al.
(author)
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Active site directed mutagenesis of 3 beta/17 beta-hydroxysteroid dehydrogenase establishes differential effects on short-chain dehydrogenase/reductase reactions
- 1997
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 36:1, s. 34-40
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Journal article (peer-reviewed)abstract
- Mutagenetic replacements uf conserved residues within the active site of the short-chain dehydrogenase/reductase (SDR) superfamily were studied using prokaryotic 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni as a model system. The results provide novel data to establish Ser138 as a member of a catalytically important ''triad'' of residues also involving Tyr151 and Lys155. A Ser --> Ala exchange at position 138 results in an almost complete (>99.9%) loss of enzymatic activity, which is not observed with a Ser --> Thr replacement. This indicates that an essential factor for catalysis is the ability of side chain 138 to form hydrogen bond interactions. Mutations in the NAD(H) binding region, in strands beta A, beta D, and adjacent turns, reveal two additional residues, Thr12 and Asn87, which are important for correct binding of the coenzyme aad with a differential effect on the reactions catalyzed. Thus, mutation of Thr12 to Ala results in a complete loss of the 3 beta-dehydrogenase activity, whereas the 3-oxoreductase activity remains unchanged. On the other hand, a T12S substitution yields a protein with unaltered catalytic constants for both reactions, revealing that a specific hydrogen bond is critical for the dehydrogenase activity. Our interpretation of the available crystal structure of 3 alpha/20 beta-HSD from Streptomyces hydrogenans suggests a hydrogen her-id in that enzyme between the Thr12 side chain and the backbone NW of Asn87 rather than the coenzyme, indicating that this hydrogen bond to the beta D strand might determine a crucial difference between the reductive and the oxidative reaction types. Similarly, mutation of Asn87 to Ala results in an 80% reduction of K-cat/K-m in the dehydrogenase direction but also unchanged 3-oxoreductase propel ties. It appears that the binding of NAD(+) to the protein is influenced by local structural changes involving strand beta D and beta A to alpha B.
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| 4. |
- Åslund, F, et al.
(author)
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Glutaredoxin-3 from Escherichia coli : Amino acid sequence, H-1 and N-15 NMR assignments, and structural analysis
- 1996
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In: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 271:12, s. 6736-6745
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Journal article (peer-reviewed)abstract
- The primary and secondary structure of glutaredoxin-3 (Grx3), a glutathione-disulfide oxidoreductase from Escherichia coli, has been determined. The amino acid sequence of Grx3 consists of 82 residues and contains a redox-active motif, Cys-Pro-Tyr-Cys, typical of the glutaredoxin family. Sequence comparison reveals a homology (33% identity) to that of glutaredoxin-1 (Grx1) from E. coli as well as to other members of the thioredoxin superfamily. in addition to the active site cysteine residues, Grx3 contains one additional cysteine (Cys(65)) corresponding to one of the two non-active site (or structural) cysteine residues present in mammalian glutaredoxins. The sequence-specific H-1 and N-15 nuclear magnetic resonance assignments of reduced Grx3 have been obtained. From a combined analysis of chemical shifts, (3)J(HN alpha) coupling constants, sequential and medium range NOEs, and amide proton exchange rates, the secondary structure of reduced Grx3 was determined and found to be very similar to that inferred from amino acid sequence comparison to homologous proteins. The consequences of the proposed structural similarity to Grx1 are that Grx3, while possessing a largely intact GSH binding cleft, would have a very different spatial distribution of charged residues, most notably surrounding the active site cysteine residues and occurring in the proposed hydrophobic protein-protein interaction area. These differences may contribute to the observed very low K-cat of Grx3 as a reductant of insulin disulfides or as a hydrogen donor for ribonucleotide reductase. Thus, despite an identical active site disulfide motif and a similar secondary structure and tertiary fold, Grx3 and Grxl display large functional differences in in vitro protein disulfide oxide-reduction reactions.
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