| 1. |
- Larsson Birgander, Pernilla, et al.
(författare)
-
Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R1
- 2005
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:15, s. 14997-15003
-
Tidskriftsartikel (refereegranskat)abstract
- Each catalytic turnover by aerobic ribonucleotide reductase requires the assembly of the two proteins, R1 (α2) and R2 (β2), to produce deoxyribonucleotides for DNA synthesis. The R2 protein forms a tight dimer, whereas the strength of the R1 dimer differs between organisms, being monomeric in mouse R1 and dimeric in Escherichia coli. We have used the known E. coli R1 structure as a framework for design of eight different mutations that affect the helices and proximal loops that comprise the dimer interaction area. Mutations in loop residues did not affect dimerization, whereas mutations in the helices had very drastic effects on the interaction resulting in monomeric proteins with very low or no activity. The monomeric N238A protein formed an interesting exception, because it unexpectedly was able to reduce ribonucleotides with a comparatively high capacity. Gel filtration studies revealed that N238A was able to dimerize when bound by both substrate and effector, a result in accordance with the monomeric R1 protein from mouse. The effects of the N238A mutation, fit well with the notion that E. coli protein R1 has a comparatively small dimer interaction surface in relation to its size, and the results illustrate the stabilization effects of substrates and effectors in the dimerization process. The identification of key residues in the dimerization process and the fact that there is little sequence identity between the interaction areas of the mammalian and the prokaryotic enzymes may be of importance in drug design, similar to the strategy used in treatment of HSV infection.
|
|
| 2. |
- Gordon, Euan, 1971, et al.
(författare)
-
Effective high-throughput overproduction of membrane proteins in Escherichia coli
- 2008
-
Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 62:1, s. 1-8
-
Tidskriftsartikel (refereegranskat)abstract
- Structural biology is increasingly reliant on elevated throughput methods for protein production. In particular, development of efficient methods of heterologous production of membrane proteins is essential. Here, we describe the heterologous overproduction of 24 membrane proteins from the human pathogen Legionella pneumophila in Escherichia coli. Protein production was performed in 0.5 ml cultures in standard 24-well plates, allowing increased throughput with minimal effort. The effect of the location of a histidine purification tag was analyzed, and the effect of decreasing the length of the N- and C-terminal extensions introduced by the Gateway cloning strategy is presented. We observed that the location and length of the purification tag significantly affected protein production levels. In addition, an auto-induction protocol for membrane protein expression was designed to enhance the overproduction efficiency such that, regardless of the construct used, much higher expression was achieved when compared with standard induction approaches such as isopropyl-β-d-thiogalactopyranoside (IPTG). All 24 targets were produced at levels exceeding 2 mg/l, with 18 targets producing at levels of 5 mg/l or higher. In summary, we have designed a fast and efficient process for the production of medically relevant membrane proteins with a minimum number of screening parameters.
|
|
| 3. |
- Hedfalk, Kristina, 1969, et al.
(författare)
-
Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
- 2008
-
Ingår i: Protein expression and purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 59:1, s. 69-78
-
Tidskriftsartikel (refereegranskat)abstract
- The causative agent of malaria, Plasmodium falciparum posses a single aquaglyceroporin (PfAQP) which represents a potential drug target for treatment of the disease. PfAQP is localized to the parasite membrane to transport water, glycerol, ammonia and possibly glycolytic intermediates. In order to enable design of inhibitors we set out to determine the 3D structure of PfAQP, where the first bottleneck to overcome is achieving high enough yield of recombinant protein. The wild type PfAQP gene was expressed to low or undetectable levels in the expression hosts, Escherichia coli and Pichia pastoris, which was assumed to be due to different genomic A+T content and different codon usage. Thus, two codon-optimized PfAQP genes were generated. The Opt-PfAQP for E. coli still did not result in high production yields, possibly due to folding problems. However, PfAQP optimized for P. pastoris was successfully expressed in P. pastoris for production and in Saccharomyces cerevisiae for functional studies. In S. cerevisiae, PfAQP mediated glycerol transport but unexpectedly water transport could not be confirmed. Following high-level membrane-localized expression in P. pastoris (estimated to 64mg PfAQP per liter cell culture) PfAQP was purified to homogeneity (18mg/L) and initial attempts at crystallization of the protein yielded several different forms.
|
|
| 4. |
- Zajicek, R. S., et al.
(författare)
-
Y25S variant of Paracoccus pantotrophus cytochrome cd1 provides insight into anion binding by d1 heme and a rare example of a critical difference between solution and crystal structures
- 2005
-
Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:28, s. 26073-26079
-
Tidskriftsartikel (refereegranskat)abstract
- Tyr25 is a ligand to the active site d1 heme in as isolated, oxidized cytochrome cd1 nitrite reductase from Paracoccus pantotrophus. This form of the enzyme requires reductive activation, a process that involves not only displacement of Tyr25 from the d1 heme but also switching of the ligands at the c heme from bis-histidinyl to His/Met. A Y25S variant retains this bis-histidinyl coordination in the crystal of the oxidized state that has sulfate bound to the d1 heme iron. This Y25S form of the enzyme does not require reductive activation, an observation previously interpreted as meaning that the presence of the phenolate oxygen of Tyr25 is the critical determinant of the requirement for activation. This interpretation now needs re-evaluation because, unexpectedly, the oxidized as prepared Y25S protein, unlike the wild type, has different heme iron ligands in solution at room temperature, as judged by magnetic circular dichroism and electron spin resonance spectroscopies, than in the crystal. In addition, the binding of nitrite and cyanide to oxidized Y25S cytochrome cd 1 is markedly different from the wild type enzyme, thus providing insight into the affinity of the oxidized d1 heme ring for anions in the absence of the steric barrier presented by Tyr25.
|
|
