| 1. |
- Aisenbrey, Christopher, et al.
(author)
-
Specific Isotope Labeling of Colicin E1 and B Channel Domains For Membrane Topological Analysis by Oriented Solid-State NMR Spectroscopy
- 2008
-
In: ChemBioChem. - : Wiley-VCH Verlagsgesellschaft. - 1439-4227 .- 1439-7633. ; 9:6, s. 944-951
-
Journal article (peer-reviewed)abstract
- An approach is presented to selectively label the methionines of the colicin E1 and B channel domains, each about 200 residues in size, and use them for oriented solid-state NMR investigations. By combining site-directed mutagenesis, bacterial overexpression in a methionine auxotroph E. coli strain and biochemical purification, quantitative amounts of the proteins for NMR structural investigations were obtained. The proteins were selectively labeled with 15N at only one, or at a few, selected sites. Multidimensional heteronuclear correlation high-resolution NMR spectroscopy and mass spectrometry were used to monitor the quality of isotopic labeling. Thereafter the proteins were reconstituted into oriented phospholipid bilayers and investigated by proton-decoupled 15N solid-state NMR spectroscopy. The colicin E1 thermolytic fragment that carries a single 15N methionine within its hydrophobic helix 9 region exhibited 15N resonances that are characteristic of helices that are oriented predominantly parallel to the membrane surface at low temperature, and a variety of alignments and conformations at room temperature. This suggests that the protein can adopt both umbrella and pen-knife conformations.
|
|
| 2. |
|
|
| 3. |
- Bergquist, Helen, 1979-, et al.
(author)
-
Structure-Specific Recognition of Friedreich’s Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives
- 2009
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 10:16, s. 2629-2637
-
Journal article (peer-reviewed)abstract
- Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)nrepeats form non-B-DNA structures, including triple helix H-DNA and higher-order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non-B-DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)nbinding by triplex-stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex-DNA-cleaving BQQ-1,10-phenanthroline (BQQ-OP) compounds. We also examined the ability of these compounds to act as structural probes for H-DNA formation within higher-order structures at pathological frataxin sequences in plasmids. DNA-complex-formation analyses with a gel-mobility-shift assay and sequence-specific probing of H-DNA-forming (GAA)nsequences by single-strand oligonucleotides and triplex-directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)nrepeats under physiologically relevant conditions.
|
|
| 4. |
- Cammenberg, Maria, et al.
(author)
-
Molecular basis for the enhanced lipase-catalyzed N-acylation of 1-phenylethanamine with methoxyacetate
- 2006
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 7:11, s. 1745-1749
-
Journal article (peer-reviewed)abstract
- One of the commercial methods for preparing enantiopure amines is lipase-catalyzed kinetic resolution, although lipases catalyze, aminolysis with only low activity. Interestingly, in 1997 Balkenhohl et al. used, ethyl methoxyacetate instead of ethyl butyrate as an acylation reagent for the aminolysis of 1-phenylethanamine and increased the reaction rate more than a 100-fold. This method has been applied to other aminolysis reactions, but the molecular basis for the enhanced rate is not understood. A moecular-modeling study of the transition-state analogue for the aminolysis showed that an interaction between the beta-oxygen atom in methoxyacetate and the amine nitrogen atom might be a key factor in the rate enhancement. Other acylation reagents, such as methyl 3-methoxypropionate and methyl 4-methoxybutyrate, were chosen to test the influence of this interaction because these molecules can be spatially arranged to have similar to that in the acylation with methyoxyacetate. The initial aminolysis rates were improved (11-fold and sixfold, respectively) compared to that with butyrate. In with 1-phenylethanol afforded the same rate with all acyl donors.
|
|
| 5. |
- Carlqvist, Peter, et al.
(author)
-
Exploring the Active-Site of a Rationally Redesigned Lipase for Catalysis of Michael-Type Additions
- 2005
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 6, s. 331-336
-
Journal article (peer-reviewed)abstract
- Michael-type additions of various thiols and alpha,beta-unsaturated carbonyl compounds were performed in organic solvent catalyzed by wild-type and a rationally redesigned mutant of Candida antarctica lipase B. The mutant locks the nucleophilic serine 105 in the active-site; this results in a changed catalytic mechanism of the enzyme. The possibility of utilizing this mutant for Michael-type additions was initially explored by quantum-chemical calculations on the reaction between acrolein and methanethiol in a model system. The model system was constructed on the basis of docking and molecular-dynamics simulations and was designed to simulate the catalytic properties of the active site. The catalytic system was explored experimentally with a range of different substrates. The k(cat) values were found to be in the range of 10(-3) to 4 min(-1), similar to the values obtained with aldolase antibodies. The enzyme proficiency was 10(7). Furthermore, the Michael-type reactions followed saturation kinetics and were confirmed to take place in the enzyme active site.
|
|
| 6. |
- Cumpstey, Ian, et al.
(author)
-
Studies of arginine-arene interactions through synthesis and evaluation of a series of galectin-binding aromatic lactose esters
- 2007
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:12, s. 1389-1398
-
Journal article (peer-reviewed)abstract
- Aromatic lactose 2-O-esters were synthesized and used to probe arene-arginine interactions with the galectin family of proteins. They were found to be low mu m inhibitors of galectin-1, -3, and -9N-terminal domain and moderate inhibitors of galectin-7, but not inhibitors of galectin-8N-terminal, which locks an arginine residue close to the critical, esterified lactose 2-O-position. Molecular modeling of galectins in complex with aromatic lactose 2-O-esters, as well as binding studies with a galectin-3 R186S mutant, confirmed that the inhibitory efficiency of the lactose 2-O-esters was due to the formation of strong interactions between the aromatic ester moieties and the arginine guanidinium groups of galectin-1 and -3. An important common feature shared by galectin-1 and -3 was that the arginines formed in-plane ion pairs with two side-chain carboxylates, which resulted in extended planar pi-electron surfaces that did not require solvation by water; these surfaces were ideal for stocking with aromatic moieties of the ligands. The results provide a basis for the design of lectin inhibitors and drugs that exploit interactions with arginine side-chains via aromatic moieties, which are involved in intramolecular protein salt bridges.
|
|
| 7. |
- Elovson Grey, Carl, et al.
(author)
-
A mass spectrometric investigation of native and oxidatively inactivated chloroperoxidase
- 2007
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 8:9, s. 1055-1062
-
Journal article (peer-reviewed)abstract
- The enzyme chloroperoxidase (CPO) found in Calclariomyces fumago is able to catalyze several stereoselective oxidation reaction by using a dean oxidant, usually hydrogen peroxide (H2O2), without the need for expensive cofactor generation. CPO's lack of operational stability however, is a major limitation for its commercial use. In the present study, a capillary-LC on-line trypsin- digestion system combined with reversed-phase chromatography and mass spectrometric detection was optimized for studying the primary sequence of CPO. Samples containing native CPO, CPO treated with H2O2, and CPO oxidatively inactivated by the use of indole and H2O2 were analyzed and compared. Three oxidized peptides were found in the samples treated with H2O2. Two additional oxidized peptides were found in the CPO samples that were completely inactivated, one of which contained an oxidized cysteine residue, Cys50, which is an essential amio acid due to its function as the axial ligand to the iron in the heme - the prosthetic group in CPO. In addition, the heme group was absent in the inactivated samples but was readily detected in other samples.
|
|
| 8. |
- Engfeldt, Torun, et al.
(author)
-
Chemical Synthesis of Triple-Labelled Three-Helix Bundle Binding Proteins for Specific Fluorescent Detection of Unlabelled Protein
- 2005
-
In: ChemBioChem. - : Wiley. - 1439-4227 .- 1439-7633. ; 6:6, s. 1043-1050
-
Journal article (peer-reviewed)abstract
- Site-specifically triple-labelled three-helix bundle affinity proteins (affibody molecules) have been produced by total chemical Synthesis. The 58 aa affinity proteins were assembled on an automated peptide synthesizer, followed by manual on-resin incorporation of three different reporter groups. An orthogonal protection strategy was developed for the site-specific introduction of 5-(2-aminethylamino)-1-nophthalenesulfonic acid (EDANS) and 6(7-nitrobenzofurazon-4-yiamino)-hexanoic acid (NBDX), constituting a donor/acceptor pair for fluorescence resonance energy transfer (FRET), and a biotin moiety, used for surface immobilization. Circular dichroism and biosensor studies of the synthetic proteins and their recombinant counterparts revealed that the synthetic proteins were folded and retained their binding specificities. The biotin-conjugated protein could be immobilized onto a streptavidin surface without loss of activity. The synthetic, doubly fluorescent-labelled affinity proteins were shown to function as fluorescent biosensors in an assay for the specific detection of unlabelled human IgG and IgA.
|
|
| 9. |
|
|
| 10. |
|
|
