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  • Grip, S., et al. (författare)
  • Engineered disulfides improve mechanical properties of recombinant spider silk
  • 2009
  • Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. - 1469-896X (Electronic) 0961-8368 (Linking) ; 18:5, s. 1012-22
  • Tidskriftsartikel (refereegranskat)abstract
    • Nature's high-performance polymer, spider silk, is composed of specific proteins, spidroins, which form solid fibers. So far, fibers made from recombinant spidroins have failed in replicating the extraordinary mechanical properties of the native material. A recombinant miniature spidroin consisting of four poly-Ala/Gly-rich tandem repeats and a nonrepetitive C-terminal domain (4RepCT) can be isolated in physiological buffers and undergoes self assembly into macrofibers. Herein, we have made a first attempt to improve the mechanical properties of 4RepCT fibers by selective introduction of AA --> CC mutations and by letting the fibers form under physiologically relevant redox conditions. Introduction of AA --> CC mutations in the first poly-Ala block in the miniature spidroin increases the stiffness and tensile strength without changes in ability to form fibers, or in fiber morphology. These improved mechanical properties correlate with degree of disulfide formation. AA --> CC mutations in the forth poly-Ala block, however, lead to premature aggregation of the protein, possibly due to disulfide bonding with a conserved Cys in the C-terminal domain. Replacement of this Cys with a Ser, lowers thermal stability but does not interfere with dimerization, fiber morphology or tensile strength. These results show that mutagenesis of 4RepCT can reveal spidroin structure-activity relationships and generate recombinant fibers with improved mechanical properties.
  • Almqvist, Jonas, et al. (författare)
  • Docking and homology modeling explain inhibition of the human vesicular glutamate transporters
  • 2007
  • Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 16:9, s. 1819-1829
  • Tidskriftsartikel (refereegranskat)abstract
    • As membrane transporter proteins, VGLUT1-3 mediate the uptake of glutamate into synaptic vesicles at presynaptic nerve terminals of excitatory neural cells. This function is crucial for exocytosis and the role of glutamate as the major excitatory neurotransmitter in the central nervous system. The three transporters, sharing 76% amino acid sequence identity in humans, are highly homologous but differ in regional expression in the brain. Although little is known regarding their three- dimensional structures, hydropathy analysis on these proteins predicts 12 transmembrane segments connected by loops, a topology similar to other members in the major facilitator superfamily, where VGLUT1-3 have been phylogenetically classified. In this work, we present a three- dimensional model for the human VGLUT1 protein based on its distant bacterial homolog in the same superfamily, the glycerol- 3-phosphate transporter from Escherichia coli. This structural model, stable during molecular dynamics simulations in phospholipid bilayers solvated by water, reveals amino acid residues that face its pore and are likely to affect substrate translocation. Docking of VGLUT1 substrates to this pore localizes two different binding sites, to which inhibitors also bind with an overall trend in binding affinity that is in agreement with previously published experimental data.
  • Andrésen, Cecilia, et al. (författare)
  • Critical biophysical properties in the Pseudomonas aeruginosa efflux gene regulator MexR are targeted by mutations conferring multidrug resistance
  • 2010
  • Ingår i: Protein Science. - : Cold Spring Harbor Laboratory Press. - 0961-8368 .- 1469-896X. ; 19:4, s. 680-692
  • Tidskriftsartikel (refereegranskat)abstract
    • The self-assembling MexA-MexB-OprM efflux pump system, encoded by the mexO operon, contributes to facile resistance of Pseudomonas aeruginosa by actively extruding multiple antimicrobials. MexR negatively regulates the mexO operon, comprising two adjacent MexR binding sites, and is as such highly targeted by mutations that confer multidrug resistance (MDR). To understand how MDR mutations impair MexR function, we studied MexR-wt as well as a selected set of MDR single mutants distant from the proposed DNA-binding helix. Although DNA affinity and MexA-MexB-OprM repression were both drastically impaired in the selected MexR-MDR mutants, MexR-wt bound its two binding sites in the mexO with high affinity as a dimer. In the MexR-MDR mutants, secondary structure content and oligomerization properties were very similar to MexR-wt despite their lack of DNA binding. Despite this, the MexR-MDR mutants showed highly varying stabilities compared with MexR-wt, suggesting disturbed critical interdomain contacts, because mutations in the DNA-binding domains affected the stability of the dimer region and vice versa. Furthermore, significant ANS binding to MexR-wt in both free and DNA-bound states, together with increased ANS binding in all studied mutants, suggest that a hydrophobic cavity in the dimer region already shown to be involved in regulatory binding is enlarged by MDR mutations. Taken together, we propose that the biophysical MexR properties that are targeted by MDR mutations stability, domain interactions, and internal hydrophobic surfaces are also critical for the regulation of MexR DNA binding.
  • Berbalk, Christoph, et al. (författare)
  • Accuracy analysis of multiple structure alignments
  • 2009
  • Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 18:10, s. 2027-2035
  • Tidskriftsartikel (refereegranskat)abstract
    • Protein structure alignment methods are essential for many different challenges in protein science, such as the determination of relations between proteins in the fold space or the analysis and prediction of their biological function. A number of different pairwise and multiple structure alignment (MStA) programs have been developed and provided to the community. Prior knowledge of the expected alignment accuracy is desirable for the user of such tools. To retrieve an estimate of the performance of current structure alignment methods, we compiled a test suite taken from literature and the SISYPHUS database consisting of proteins that are difficult to align. Subsequently, different MStA programs were evaluated regarding alignment correctness and general limitations. The analysis shows that there are large differences in the success between the methods in terms of applicability and correctness. The latter ranges from 44 to 75% correct core positions. Taking only the best method result per test case this number increases to 84%. We conclude that the methods available are applicable to difficult cases, but also that there is still room for improvements in both, practicability and alignment correctness. An approach that combines the currently available methods supported by a proper score would be useful. Until then, a user should not rely on just a single program.
  • Bergkvist, Anders, et al. (författare)
  • 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
  • Ingår i: Protein Science. - : John Wiley & Sons. - 0961-8368 .- 1469-896X. ; 10:12, s. 2623-2626
  • 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.
  • Berglund, Lisa, et al. (författare)
  • The epitope space of the human proteome
  • 2008
  • Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 17:4, s. 606-613
  • Tidskriftsartikel (refereegranskat)abstract
    • In the post-genome era, there is a great need for protein-specific affinity reagents to explore the human proteome. Antibodies are suitable as reagents, but generation of antibodies with low cross-reactivity to other human proteins requires careful selection of antigens. Here we show the results from a proteomewide effort to map linear epitopes based on uniqueness relative to the entire human proteome. The analysis was based on a sliding window sequence similarity search using short windows (8, 10, and 12 amino acid residues). A comparison of exact string matching (Hamming distance) and a heuristic method (BLAST) was performed, showing that the heuristic method combined with a grid strategy allows for whole proteome analysis with high accuracy and feasible run times. The analysis shows that it is possible to find unique antigens for a majority of the human proteins, with relatively strict rules involving low sequence identity of the possible linear epitopes. The implications for human antibody-based proteomics efforts are discussed.
  • Carnrot, Cecilia, et al. (författare)
  • Mechanisms of substrate selectivity for Bacillus anthracis thymidylate kinase
  • 2008
  • Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 17:9, s. 1486-1493
  • Tidskriftsartikel (refereegranskat)abstract
    • Bacillus anthracis is well known in connection with biological warfare. The search for new drug targets and antibiotics is highly motivated because of upcoming multiresistant strains. Thymidylate kinase is an ideal target since this enzyme is at the junction of the de novo and salvage synthesis of dTTP, an essential precursor for DNA synthesis. Here the expression and characterization of thymidylate kinase from B. anthracis (Ba-TMPK) is presented. The enzyme phosphorylated deoxythymidine-5'-monophosphate (dTMP) efficiently with K-m and V-max values of 33 mu M and 48 mu mol mg(-1) min(-1), respectively. The efficiency of deoxyuridine-5'-monophosphate phosphorylation was; similar to 10% of that of dTMP. Several dTMP analogs were tested, and D-FMAUMP (2'-fluoroarabinosyl-5-methyldeoxyuridine-5'- monophosphate) was selectively phosphorylated with an efficiency of 172% of that of D-dTMP, but L-FMAUMP was a poor substrate as were 5-fluorodeoxyuridine-5'-monophosphate (5FdUMP) and 2',3'-dideoxy-2',3'-didehydrothymidine-5'-monophosphate (d4TMP). No activity could be detected with 3'-azidothymidine-5'-monophosphate (AZTMP). The corresponding nucleosides known as efficient anticancer and antiviral compounds were also tested, and D-FMAU was a strong inhibitor with an IC50 value of 10 mu M, while other nucleosides-L-FMAU, dThd, 5-FdUrd, d4T, and AZT, and 2'-arabinosylthymidine-were poor inhibitors. A structure model was built for Ba-TMPK based on the Staphylococcus aureus TMPK structure. Docking with various substrates suggested mechanisms explaining the differences in substrate selectivity of the human and the bacterial TMPKs. These results may serve as a start point for development of new antibacterial agents.
  • De Marothy, Minttu T., et al. (författare)
  • Marginally hydrophobic transmembrane alpha-helices shaping membrane protein folding
  • 2015
  • Ingår i: Protein Science. - 0961-8368 .- 1469-896X. ; 24:7, s. 1057-1074
  • Forskningsöversikt (refereegranskat)abstract
    • Cells have developed an incredible machinery to facilitate the insertion of membrane proteins into the membrane. While we have a fairly good understanding of the mechanism and determinants of membrane integration, more data is needed to understand the insertion of membrane proteins with more complex insertion and folding pathways. This review will focus on marginally hydrophobic transmembrane helices and their influence on membrane protein folding. These weakly hydrophobic transmembrane segments are by themselves not recognized by the translocon and therefore rely on local sequence context for membrane integration. How can such segments reside within the membrane? We will discuss this in the light of features found in the protein itself as well as the environment it resides in. Several characteristics in proteins have been described to influence the insertion of marginally hydrophobic helices. Additionally, the influence of biological membranes is significant. To begin with, the actual cost for having polar groups within the membrane may not be as high as expected; the presence of proteins in the membrane as well as characteristics of some amino acids may enable a transmembrane helix to harbor a charged residue. The lipid environment has also been shown to directly influence the topology as well as membrane boundaries of transmembrane helices-implying a dynamic relationship between membrane proteins and their environment.
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