| 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. |
- Brocca, Stefania, et al.
(författare)
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Mutants provide evidence of the importance of glycosydic chains in the activation of lipase 1 from Candida rugosa
- 2000
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 9:5, s. 985-990
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Tidskriftsartikel (refereegranskat)abstract
- Sequence analysis of Candida rugosa lipase 1 (LIP1) predicts the presence of three N-linked glycosylation sites at asparagine 291, 314, 351. To investigate the relevance of sugar chains in the activation and stabilization of LIP1, we directed site mutagenesis to replace the above mentioned asparagine with glutamine residues. Comparison of the activity of mutants with that of the wild-type (wt) lipase indicates that both 314 and 351 Asn to Gln substitutions influence, although at a different extent, the enzyme activity both in hydrolysis and esterification reactions, but they do not alter the enzyme water activity profiles in organic solvents or temperature stability. Introduction of Gln to replace Asn35 is likely to disrupt a stabilizing interaction between the sugar chain and residues of the inner side of the lid in the enzyme active conformation. The effect of deglycosylation at position 314 is more difficult to explain and might suggest a more general role of the sugar moiety for the structural stability of lipase 1. Conversely, Asn291Gln substitution does not affect' the lipolytic or the esterase activity of the mutant that behaves essentially as the wt enzyme. This observation supports the hypothesis that changes in activity of Asn314Gln and Asn351Gln mutants are specifically due to deglycosylation.
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| 3. |
- Casbarra, A, et al.
(författare)
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Conformational analysis of HAMLET, the folding variant of human alpha-lactalbumin associated with apoptosis
- 2004
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 13:5, s. 1322-1330
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Tidskriftsartikel (refereegranskat)abstract
- A combination of hydrogen/deuterium (H/D) exchange and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the conformation in solution of HAMLET, the folding variant of human alpha-lactalbumin, complexed to oleic acid, that induces apoptosis in tumor and immature cells. Although near- and far-UV CD and fluorescence spectroscopy were not able to discriminate between HAMLET and apo-alpha-lactalbumin, H/D exchange experiments clearly showed that they correspond to two distinct conformational states, with HAMLET incorporating a greater number of deuterium atoms than the apo and holo forms. Complementary proteolysis experiments revealed that HAMLET and apo are both accessible to proteases in the P-domain but showed substantial differences in accessibility to proteases at specific sites. The overall results indicated that the conformational changes associated with the release of Ca2+ are not sufficient to induce the HAMLET conformation. Metal depletion might represent the first event to produce a partial unfolding in the beta-domain of a-lactalbumin, but some more unfolding is needed to generate the active conformation HAMLET, very likely allowing the protein to bind the C18:1 fatty acid moiety. On the basis of these data, a putative binding site of the oleic acid, which stabilizes the HAMLET conformation, is proposed.
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| 4. |
- Gottschalk, Michael, et al.
(författare)
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Protein self-association in solution: the bovine beta-lactoglobulin dimer and octamer
- 2003
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 12:11, s. 2404-2411
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Tidskriftsartikel (refereegranskat)abstract
- We have used proton magnetic relaxation dispersion (MRD) to study the self-association of bovine ß-lactoglobulin variant A (BLG-A) as a function of temperature at pH 4.7 (dimer–octamer equilibrium) and as a function of NaCl concentration at pH 2.5 (monomer–dimer equilibrium). The MRD method identifies coexisting oligomers from their rotational correlation times and determines their relative populations from the associated dispersion amplitudes. From MRD-derived correlation times and hydrodynamic model calculations, we confirm that BLG-A dimers associate to octamers below room temperature. The tendency for BLG-A dimers to assemble into octamers is found to be considerably weaker than in previous light scattering studies in the presence of buffer salt. At pH 2.5, the MRD data are consistent with an essentially complete transition from monomers in the absence of salt to dimers in 1 M NaCl. Because of an interfering relaxation dispersion from nanosecond water exchange, we cannot determine the oligomer populations at intermediate salt concentrations. This nanosecond dispersion may reflect intersite exchange of water molecules trapped inside the large binding cavity of BLG-A.
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| 5. |
- Gustavsson, Niklas, et al.
(författare)
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Substitution of conserved methionines by leucines in chloroplast small heat shock protein results in loss of redox-response but retained chaperone-like activity
- 2001
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Ingår i: Protein Science. - 1469-896X. ; 10:9, s. 1785-1793
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Tidskriftsartikel (refereegranskat)abstract
- During evolution of land plants, a specific motif occurred in the N-terminal domain of the chloroplast-localized small heat shock protein, Hsp21: a sequence with highly conserved methionines, which is predicted to form an amphipathic -helix with the methionines situated along one side. The functional role of these conserved methionines is not understood. We have found previously that treatment, which causes methionine sulfoxidation in Hsp21, also leads to structural changes and loss of chaperone-like activity. Here, mutants of Arabidopsis thaliana Hsp21 protein were created by site-directed mutagenesis, whereby conserved methionines were substituted by oxidation-resistant leucines. Mutants lacking the only cysteine in Hsp21 were also created. Protein analyses by nondenaturing electrophoresis, size exclusion chromatography, and circular dichroism proved that sulfoxidation of the four highly conserved methionines (M49, M52, M55, and M59) is responsible for the oxidation-induced conformational changes in the Hsp21 oligomer. In contrast, the chaperone-like activity was not ultimately dependent on the methionines, because it was retained after methionine-to-leucine substitution. The functional role of the conserved methionines in Hsp21 may be to offer a possibility for redox control of chaperone-like activity and oligomeric structure dynamics.
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| 6. |
- Hammarström, Martin, et al.
(författare)
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Rapid screening for improved solubility of small human proteins produced as fusio proteins in Escherichia coli.
- 2002
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 11:2, s. 313-321
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Tidskriftsartikel (refereegranskat)abstract
- A prerequisite for structural genomics and related projects is to standardize the process of gene overexpression and protein solubility screening to enable automation for higher throughput. We have tested a methodology to rapidly subclone a large number of human genes and screen these for expression and protein solubility in Escherichia coli. The methodology, which can be partly automated, was used to compare the effect of six different N-terminal fusion proteins and an N-terminal 6*His tag. As a realistic test set we selected 32 potentially interesting human proteins with unknown structures and sizes suitable for NMR studies. The genes were transferred from cDNA to expression vectors using subcloning by recombination. The subcloning yield was 100% for 27 (of 32) genes for which a PCR fragment of correct size could be obtained. Of these, 26 genes (96%) could be overexpressed at detectable levels and 23 (85%) are detected in the soluble fraction with at least one fusion tag. We find large differences in the effects of fusion protein or tag on expression and solubility. In short, four of seven fusions perform very well, and much better than the 6*His tag, but individual differences motivate the inclusion of several fusions in expression and solubility screening. We also conclude that our methodology and expression vectors can be used for screening of genes for structural studies, and that it should be possible to obtain a large fraction of all NMR-sized and nonmembrane human proteins as soluble fusion proteins in E. coli.
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| 7. |
- Jain, Nitin U., et al.
(författare)
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Distance mapping of protein-binding sites using spin-labeled oligosaccharide ligands
- 2001
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Ingår i: Protein Science. - : Wiley. - 1469-896X .- 0961-8368. ; 10:11, s. 2393-2400
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Tidskriftsartikel (refereegranskat)abstract
- The binding of a nitroxide spin-labeled analog of N-acetyllactosamine to galectin-3, a mammalian lectin of 26 kD size, is studied to map the binding sites of this small oligosaccharide on the protein surface. Perturbation of intensities of cross-peaks in the (15)N heteronuclear single quantum coherence (HSQC) spectrum of full-length galectin-3 owing to the bound spin label is used qualitatively to identify protein residues proximate to the binding site for N-acetyllactosamine. A protocol for converting intensity measurements to a more quantitative determination of distances between discrete protein amide protons and the bound spin label is then described. This protocol is discussed as part of a drug design strategy in which subsequent perturbation of chemical shifts of distance mapped amide cross-peaks can be used effectively to screen a library of compounds for other ligands that bind to the target protein at distances suitable for chemical linkage to the primary ligand. This approach is novel in that it bypasses the need for structure determination and resonance assignment of the target protein.
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