| 1. |
- Anderbrant, O., et al.
(författare)
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Electrophysiological and morphological characteristics of pheromone receptors in male pine sawflies, Diprion pini (Hymenoptera : Diprionidae), and behavioural response to some compounds
- 1995
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Ingår i: Journal of Insect Physiology. - : Elsevier BV. - 0022-1910. ; 41:5, s. 395-401
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Tidskriftsartikel (refereegranskat)abstract
- The morphology and physiology of pheromone receptors on the antennae of male pine sawflies, Diprion pini L., were investigated. Using scanning electron microscopy, five sensillar types were recognized. The type shown to be pheromone sensitive has a long (50-70 μm) cuticular hair, is single-walled, and is innervated by 8 or 9 sensory cells as revealed by transmission electron microscopy. Electroantennography (EAG) showed similar activity of the acetate and propionate of (2S,3R,7R)-3,7-dimethyl-2-tridecanol, precursor of the main constituent of the female-produced sex pheromone. No other isomer induced any significant response. Single-sensillum recordings confirmed the results of the EAG, and also showed that several neurons were excited by the active compound. EAG recordings and combined gas chromatographic-electroantennographic detection indicated that esters of three 3,7-dimethyl-2-pentadecanol (diprionol) isomers were active, but field tests could not demonstrate any behavioural effect. Diprionol esters are used as sex pheromones by all other pine sawflies investigated so far, and D. pini is thus the first diprionid species shown to use a different sex pheromone.
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| 2. |
- Evenäs, Johan, et al.
(författare)
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NMR studies of the E140Q mutant of the carboxy-terminal domain of calmodulin reveal global conformational exchange in the Ca2+-saturated state
- 1997
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 36:12, s. 3448-3457
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Tidskriftsartikel (refereegranskat)abstract
- In the present investigation, the Ca2+ activation of the C-terminal domain of bovine calmodulin and the effects of replacing the bidentate Ca2+-coordinating glutamic acid residue in the 12th and last position of loop IV with a glutamine are studied by NMR spectroscopy. The mutation E140Q results in sequential Ca2+ binding in this domain and has far-reaching effects on the structure of (Ca2+)2 TR2C, thereby providing further evidence for the critical role of this glutamic acid residue for the Ca2+- induced conformational change of regulatory EF-hand proteins. Analyses of the NOESY spectra of the mutant under Ca2+-saturated conditions, such that 97% of the protein is in the (Ca2+)2 form, revealed two sets of mutually exclusive NOEs. One set of NOEs is found to be consistent with the closed structure observed in the apo state of the C-terminal domain of the wild- type protein, while the other set supports the open structure observed in the Ca2+-saturated state. In addition, several residues in the hydrophobic core exhibit broadened resonances. We conclude that the (Ca2+)2 form of the mutant experiences a global conformational exchange between states similar to the closed and open conformations of the C-terminal domain of wild-type calmodulin. A population of 65 ± 15% of the open conformation and an exchange rate of (1-7) x 104 s-1 were estimated from the NMR data and the chemical shifts of the wild-type protein. From a Ca2- titration of the 15N-labeled mutant, the macroscopic binding constants (log(K1) = 4.9 ± 0.3 and log(K2) = 3.15 ± 0.10] and the inherent chemical shifts of the intermediate (Ca2+)1 form of the mutant were determined using NMR. Valuable information was also provided on the mechanism of the Ca2+ activation and the roles of the structural elements in the two Ca2+- binding events. Comparison with the wild-type protein indicates that the (Ca2+)1 conformation of the mutant is essentially closed but that some rearrangement of the empty loop IV toward the Ca2+-bound form has occurred.
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| 3. |
- Wu, Wenqi, et al.
(författare)
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Discrimination among pheromone component blends by interneurons in male antennal lobes of two populations of the turnip moth, Agrotis segetum
- 1996
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424. ; 93:15, s. 8022-8027
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Tidskriftsartikel (refereegranskat)abstract
- A difference in female pheromone production and male behavioral response has previously been found in two populations of the turnip moth, Agrotis segetum, originating from Sweden and Zimbabwe, respectively. In this study, we investigated the pheromone response of antennal lobe interneurons of males of the two populations by intracellular recordings, stimulating with single pheromone components and various inter- and intra-populational pheromone blends. Three major physiological types of antennal lobe neurons were established in the two populations according to their responses to different stimuli. One type responded broadly to almost all the stimuli tested. The second type responded selectively to some of the single components and blends. The third type did not respond to any single components but did respond to certain blends. Furthermore, some neurons of the second and third type recognized strain specific differences in ratios between pheromone components. Both projection neurons and local interneurons were found among these three types. Two pheromone responding bilateral projection neurons are reported for the first time in this paper.
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| 4. |
- Carlsson, Jonas, et al.
(författare)
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Affinity precipitation and site-specific immobilization of proteins carrying polyhistidine tails
- 1996
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Ingår i: Biotechnology and Bioengineering. - 0006-3592. ; 51:2, s. 8-221
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Tidskriftsartikel (refereegranskat)abstract
- Proteins carrying genetically attached polyhistidine tails have been purified using affinity precipitation with metal chelates. DNA fragments encoding four or five histidine residues have been genetically fused to the oligomeric enzymes lactate dehydrogenase (Bacillus stearothermophilus), beta-glucoronidase (Escherichia coli), and galactose dehydrogenase (Pseudomonas fluorescens) as well as to the monomeric protein A (Staphylococcus aureus). The chimeric genes were subsequently expressed in E. coli. The engineered enzymes were successfully purified from crude protein solutions using ethylene glycolbis (beta-aminoethyl) tetraacetic acid (EGTA) charged with Zn(2+) as precipitant, whereas protein A, carrying only one attached histidine tail, did not precipitate. However, all of the engineered proteins could be purified on immobilized metal affinity chromatography (IMAC) columns loaded with Zn(2+). The potential of using the same histidine tails for site-specific immobilization of proteins was also investigated. The enzymes were all catalytically active when immobilized on IMAC gels. For instance, immobilized lactate dehydrogenase, carrying tails composed of four histidine residues, displaced 83% of the soluble enzyme activity.
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| 5. |
- Al-Karadaghi, Salam, et al.
(författare)
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Crystal structure of ferrochelatase: the terminal enzyme in heme biosynthesis
- 1997
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Ingår i: Structure. - 0969-2126. ; 5:11, s. 1501-1510
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: The metallation of closed ring tetrapyrroles resulting in the formation of hemes, chlorophylls and vitamin B12 is catalyzed by specific enzymes called chelatases. Ferrochelatase catalyzes the terminal step in heme biosynthesis by inserting ferrous ion into protoporphyrin IX by a mechanism that is poorly understood. Mutations in the human gene for ferrochelatase can result in the disease erythropoietic protoporphyria, and a further understanding of the mechanism of this enzyme is therefore of clinical interest. No three-dimensional structure of a tetrapyrrole metallation enzyme has been available until now. RESULTS: The three-dimensional structure of Bacillus subtilis ferrochelatase has been determined at 1.9 A resolution by the method of multiple isomorphous replacement. The structural model contains 308 of the 310 amino acid residues of the protein and 198 solvent molecules. The polypeptide is folded into two similar domains each with a four-stranded parallel beta sheet flanked by alpha helices. Structural elements from both domains build up a cleft, which contains several amino acid residues that are invariant in ferrochelatases from different organisms. In crystals soaked with gold and cadmium salt solutions, the metal ion was found to be coordinated to the conserved residue His 183, which is located in the cleft. This histidine residue has previously been suggested to be involved in ferrous ion binding. CONCLUSIONS: Ferrochelatase seems to have a structurally conserved core region that is common to the enzyme from bacteria, plants and mammals. We propose that porphyrin binds in the identified cleft; this cleft also includes the metal-binding site of the enzyme. It is likely that the structure of the cleft region will have different conformations upon substrate binding and release.
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| 6. |
- Andersson, Dick
(författare)
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Conformational Studies of Protein Structure and Dynamics
- 1999
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The protein folding problem is one of the most important issues to be solved in the field of molecular biology. The subject of this thesis mainly deals with various aspects of the folding process.In Paper I, near-UV CD kinetic measurements on mutants, in which one tryptophan (Trp) residue had been replaced, were performed to probe the development of asymmetric environments around specific Trp residues during the refolding of human carbonic anhydrase II (HCA II). The development of the individual (Trp) CD spectra during refolding was obtained by subtracting the CD spectrum of the mutant lacking one Trp from that of HCA II at different time points. The same method was used for the particular Trp residues to obtain the kinetic CD traces monitored at a specific wavelength (270 nm). Three Trp residues were analyzed, each probing different structural regions of the native structure. The investigated Trp residues develop their native CD bands at different rates, showing that formation of native-like tertiary structure is occurring with varying rates indifferent regions of the protein.The same approach was applied to the extracellular domain of human tissue factor (sTF), which contains four Trp residues (Paper II). The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated, including the far-UV region. This leads to uncertain predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp-free CD spectrum. The kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.In Paper III, the role of the metal ion cofactor for the refolding of bovine carbonic anhydrase II (BCA II) was studied from the molten globule to the native state. Refolding was possible to achieve by mere addition of the metal ion to the apomolten-globule, because the apoenzyme was less stable than the holoenzyme. The cofactor-effected refolding can be summarized as follows: 1) initially, the metal ion binds to the molten globule; 2) compaction of the metal-binding site region is then induced by the metal ion binding; 3) a functioning active center is formed; 4) finally, the native tertiary structure is generated in the outer parts of the protein.In paper IV the aim was to determine the nature of the tetramer contact of human extracellular superoxide dismutase (hEC-SOD). We chose a strategy in which we mapped the subunit interaction interface by studying effects of twelve different mutations in the N-terminal domain fused to HCA II. The results show that the hydrophobic side of a predicted amphiphatic a-helix (formed by residues 14-32) in the N-terminal domain is essential for the subunit interaction.
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| 7. |
- Berndt, Kurt D, et al.
(författare)
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Conformational sampling by NMR solution structures calculated with the program DIANA evaluated by comparison with long-time molecular dynamics calculations in explicit water
- 1996
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Ingår i: Proteins. - 0887-3585 .- 1097-0134. ; 24, s. 304-313
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Tidskriftsartikel (refereegranskat)abstract
- The NMR solution structure of bovine pancreatic trypsin inhibitor (BPTI) obtained by distance geometry calculations with the program DIANA is compared with groups of conformers generated by molecular dynamics (MD) simulations in explicit water at ambient temperature and pressure. The MD simulations started from a single conformer and were free or restrained either by the experimental NOE distance restraints or by time-averaged restraints; the groups of conformers were collected either in 10 ps intervals during 200 ps periods of simulation, or in 50 ps intervals during a 1 ns period of simulation. Overall, these comparisons show that the standard protein structure determination protocol with the program DIANA provides a picture of the protein structure that is in agreement with MD simulations using "realistic" potential functions over a nanosecond timescale. For well-constrained molecular regions there is a trend in the free MD simulation of duration 1 ns that the sampling of the conformation space is slightly increased relative to the DIANA calculations. In contrast, for surface-exposed side-chains that are less extensively constrained by the NMR data, the DIANA conformers tend to sample larger regions of conformational space than conformers selected from any of the MD trajectories. Additional insights into the behavior of surface side-chains come from comparison of the MD runs of 200 ps or 1 ns duration. In this time range the sampling of conformation space by the protein surface depends strongly on the length of the simulation, which indicates that significant side-chain transitions occur on the nanosecond timescale and that much longer simulations will be needed to obtain statistically significant data on side-chain dynamics.
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| 8. |
- Bernier-Villamor, V, et al.
(författare)
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Crystallization and preliminary X-ray diffraction of Trypanosoma cruzi dUTPase
- 1999
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D55, s. 528-530
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Tidskriftsartikel (refereegranskat)abstract
- Crystals of Trypanosoma cruzi dUTPase have been grown. Two different morphologies are observed, depending on the molecular weight of the PEG used as precipitating agent in the mother liquor, both having a hexagonal unit cell with similar dimensions. Complete X-ray diffraction data have been collected to low resolution for one of the forms. The space group is P6322, with unit-cell dimensions a = 134.15, c = 147.05 Å. Peaks in the self-rotation function and the solvent content are consistent with two molecules of dUTPase per asymmetric unit.
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| 9. |
- Brunne, R M, et al.
(författare)
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Structure and internal dynamics of the bovine pancreatic trypsin inhibitor in aqueous solution from long-time molecular dynamics simulations
- 1995
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Ingår i: Proteins. - : Wiley. - 0887-3585 .- 1097-0134. ; 23:1, s. 49-62
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Tidskriftsartikel (refereegranskat)abstract
- Structural and dynamic properties of bovine pancreatic trypsin inhibitor (BPTI) in aqueous solution are investigated using two molecular dynamics (MD) simulations: one of 1.4 ns length and one of 0.8 ns length in which atom-atom distance bounds derived from NMR spectroscopy are included in the potential energy function to make the trajectory satisfy these experimental data more closely. The simulated properties of BPTI are compared with crystal and solution structures of BPTI, and found to be in agreement with the available experimental data. The best agreement with experiment was obtained when atom-atom distance restraints were applied in a time-averaged manner in the simulation. The polypeptide segments found to be most flexible in the MD simulations coincide closely with those showing differences between the crystal and solution structures of BPTI.
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| 10. |
- Dauter, Z, et al.
(författare)
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The Refined Structure of dUTPase from Escherichia coli
- 1998
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Ingår i: Acta Crystallographica. Section D: Biological Crystallography. - 1399-0047. ; D54:5, s. 735-749
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Tidskriftsartikel (refereegranskat)abstract
- Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase, E.C. 3.6.1.23) catalyzes the hydrolysis of dUTP to dUMP and pyrophosphate and is involved in nucleotide metabolism and DNA synthesis. A crystal of the recombinant E. coli enzyme, precipitated from polyethylene glycol mixtures in the presence of succinate at pH 4.2, was used to collect synchrotron diffraction data to 1.9 Å resolution, in space group R3, a = b = 86.62, c = 62.23 Å. Mercury and platinum derivative data were collected at wavelengths to optimize the anomalous contribution. The resulting 2.2 Å MIRAS phases differed from the final set by 40° on average and produced an excellent map which was easy to interpret. The model contains 132 water molecules and refined to an R value of 13.7%. 136 residues have clear electron density out of 152 expected from the gene sequence. The 16 C-terminal residues are presumably disordered in the crystal lattice. The monomer is a `jelly-roll' type, containing mostly -sheet and only one short helix. The molecule is a tight trimer. A long C-terminal arm extends from one subunit and encompasses the next one within the trimer contributing to its -sheet. Conserved sequence motifs common among dUTPases, previously suggested to compose the active site and confirmed in a recent study of the dUDP complex, are located at subunit-subunit interfaces along the threefold axis, in parts of the -sheet and in loop regions. A similar molecular architecture has recently been found in two other trimeric dUTPases.
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