| 1. |
- Larsson Birgander, Pernilla, et al.
(författare)
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Nucleotide-dependent formation of catalytically competent dimers from engineered monomeric ribonucleotide reductase protein R1
- 2005
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:15, s. 14997-15003
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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.
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| 2. |
- Bestas, Burcu, et al.
(författare)
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A Type II-B Cas9 nuclease with minimized off-targets and reduced chromosomal translocations in vivo
- 2023
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Ingår i: NATURE COMMUNICATIONS. - 2041-1723. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes Cas9 (SpCas9) and derived enzymes are widely used as genome editors, but their promiscuous nuclease activity often induces undesired mutations and chromosomal rearrangements. Several strategies for mapping off-target effects have emerged, but they suffer from limited sensitivity. To increase the detection sensitivity, we develop an off-target assessment workflow that uses Duplex Sequencing. The strategy increases sensitivity by one order of magnitude, identifying previously unknown SpCas9's off-target mutations in the humanized PCSK9 mouse model. To reduce off-target risks, we perform a bioinformatic search and identify a high-fidelity Cas9 variant of the II-B subfamily from Parasutterella secunda (PsCas9). PsCas9 shows improved specificity as compared to SpCas9 across multiple tested sites, both in vitro and in vivo, including the PCSK9 site. In the future, while PsCas9 will offer an alternative to SpCas9 for research and clinical use, the Duplex Sequencing workflow will enable a more sensitive assessment of Cas9 editing outcomes. SpCas9 unintended editing is a major concern. Here the authors report an off-target method using Duplex Sequencing with increased sensitivity for Cas9 mutation detection; they also identify a Cas9 variant of the II-B subfamily with intrinsic high fidelity (PsCas9) and see improved specificity.
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| 3. |
- Bigalke, Janna M., et al.
(författare)
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Cryo-EM structure of the activated RET signaling complex reveals the importance of its cysteine-rich domain
- 2019
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Ingår i: Science Advances. - : American Association for the Advancement of Science (AAAS). - 2375-2548. ; 5:7
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Tidskriftsartikel (refereegranskat)abstract
- Signaling through the receptor tyrosine kinase RET is essential during normal development. Both gain- and loss-of-function mutations are involved in a variety of diseases, yet the molecular details of receptor activation have remained elusive. We have reconstituted the complete extracellular region of the RET signaling complex together with Neurturin (NRTN) and GFR alpha 2 and determined its structure at 5.7-angstrom resolution by cryo-EM. The proteins form an assembly through RET-GFR alpha 2 and RET-NRTN interfaces. Two key interaction points required for RET extracellular domain binding were observed: (i) the calcium-binding site in RET that contacts GFR alpha 2 domain 3 and (ii) the RET cysteine-rich domain interaction with NRTN. The structure highlights the importance of the RET cysteine-rich domain and allows proposition of a model to explain how complex formation leads to RET receptor dimerization and its activation. This provides a framework for targeting RET activity and for further exploration of mechanisms underlying neurological diseases.
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| 4. |
- Flenady, Vicki, et al.
(författare)
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Stillbirths : recall to action in high-income countries.
- 2016
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Ingår i: The Lancet. - 0140-6736 .- 1474-547X. ; 387:10019, s. 691-702
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Tidskriftsartikel (refereegranskat)abstract
- Variation in stillbirth rates across high-income countries and large equity gaps within high-income countries persist. If all high-income countries achieved stillbirth rates equal to the best performing countries, 19,439 late gestation (28 weeks or more) stillbirths could have been avoided in 2015. The proportion of unexplained stillbirths is high and can be addressed through improvements in data collection, investigation, and classification, and with a better understanding of causal pathways. Substandard care contributes to 20-30% of all stillbirths and the contribution is even higher for late gestation intrapartum stillbirths. National perinatal mortality audit programmes need to be implemented in all high-income countries. The need to reduce stigma and fatalism related to stillbirth and to improve bereavement care are also clear, persisting priorities for action. In high-income countries, a woman living under adverse socioeconomic circumstances has twice the risk of having a stillborn child when compared to her more advantaged counterparts. Programmes at community and country level need to improve health in disadvantaged families to address these inequities.
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| 5. |
- Gordon, Euan, 1971, et al.
(författare)
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Effective high-throughput overproduction of membrane proteins in Escherichia coli
- 2008
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 62:1, s. 1-8
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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.
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| 6. |
- Gutgsell, Aspen Rene, et al.
(författare)
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Biosensor-Enabled Deconvolution of the Avidity-Induced Affinity Enhancement for the SARS-CoV-2 Spike Protein and ACE2 Interaction
- 2022
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 94:2, s. 1187-1194
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Tidskriftsartikel (refereegranskat)abstract
- Avidity is an effective and frequent phenomenon employed by nature to achieve extremely high-affinity interactions. As more drug discovery efforts aim to disrupt protein-protein interactions, it is becoming increasingly common to encounter systems that utilize avidity effects and to study these systems using surface-based technologies, such as surface plasmon resonance (SPR) or biolayer interferometry. However, heterogeneity introduced from multivalent binding interactions complicates theanalysis of the resulting sensorgram. A frequently applied practice is to fit the data based on a 1:1 binding model, and if the fit does not describe the data adequately, then the experimental setup is changed to favor a 1:1 binding interaction. This reductionistic approach is informative but not always biologically relevant. Therefore, we aimed to develop an SPR-based assay that would reduce the heterogeneity to enable the determination of the kinetic rate constants for multivalent binding interactions using the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein and the human receptor angiotensin-converting enzyme 2 (ACE2) as a model system. We employed a combinatorial approach to generate a sensor surface that could distinguish between monovalent and multivalent interactions. Using advanced data analysis algorithms to analyze the resulting sensorgrams, we found that controlling the surface heterogeneity enabled the deconvolution of theavidity-induced affinity enhancement for the SARS-CoV-2 spike protein and ACE2 interaction.
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| 7. |
- Hedfalk, Kristina, 1969, et al.
(författare)
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Production, characterization and crystallization of the Plasmodium falciparum aquaporin.
- 2008
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Ingår i: Protein expression and purification. - : Elsevier BV. - 1096-0279 .- 1046-5928. ; 59:1, s. 69-78
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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.
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| 8. |
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| 9. |
- Kasrayan, Alex, et al.
(författare)
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Enhancement by effectors and substrate nucleotides of R1-R2 interactions in Escherichia coli class Ia ribonucleotide reductase.
- 2004
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Ingår i: J Biol Chem. - 0021-9258. ; 279:30, s. 31050-7
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Ribonucleotide reductases are a family of essential enzymes that catalyze the reduction of ribonucleotides to their corresponding deoxyribonucleotides and provide cells with precursors for DNA synthesis. The different classes of ribonucleotide reductase are distinguished based on quaternary structures and enzyme activation mechanisms, but the components harboring the active site region in each class are evolutionarily related. With a few exceptions, ribonucleotide reductases are allosterically regulated by nucleoside triphosphates (ATP and dNTPs). We have used the surface plasmon resonance technique to study how allosteric effects govern the strength of quaternary interactions in the class Ia ribonucleotide reductase from Escherichia coli, which like all class I enzymes has a tetrameric alpha(2) beta(2) structure. The component alpha(2)called R1 harbors the active site and two types of binding sites for allosteric effector nucleotides, whereas the beta(2) component called R2 harbors the tyrosyl radical necessary for catalysis. Our results show that only the known allosteric effector nucleotides, but not non-interacting nucleotides, promote a specific interaction between R1 and R2. Interestingly, the presence of substrate together with allosteric effector nucleotide strengthens the complex 2-3 times with a similar free energy change as the mutual allosteric effects of substrate and effector nucleotide binding to protein R1 in solution experiments. The dual allosteric effects of dATP as positive allosteric effector at low concentrations and as negative allosteric effector at high concentrations coincided with an almost 100-fold stronger R1-R2 interaction. Based on the experimental setup, we propose that the inhibition of enzyme activity in the E. coli class Ia enzyme occurs in a tight 1:1 complex of R1 and R2. Most intriguingly, we also discovered that thioredoxin, one of the physiological reductants of ribonucleotide reductases, enhances the R1-R2 interaction 4-fold.
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| 10. |
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| 11. |
- Zajicek, R. S., et al.
(författare)
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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
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 280:28, s. 26073-26079
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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.
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