| 1. |
- Diamanti, Riccardo, et al.
(författare)
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Comparative structural analysis provides new insights into the function of R2-like ligand-binding oxidase
- 2022
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Ingår i: FEBS Letters. - : John Wiley & Sons. - 0014-5793 .- 1873-3468. ; 596:12, s. 1600-1610
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Tidskriftsartikel (refereegranskat)abstract
- R2-like ligand-binding oxidase (R2lox) is a ferritin-like protein that harbours a heterodinuclear manganese–iron active site. Although R2lox function is yet to be established, the enzyme binds a fatty acid ligand coordinating the metal centre and catalyses the formation of a tyrosine–valine ether cross-link in the protein scaffold upon O2 activation. Here, we characterized the ligands copurified with R2lox by mass spectrometry-based metabolomics. Moreover, we present the crystal structures of two new homologs of R2lox, from Saccharopolyspora erythraea and Sulfolobus acidocaldarius, at 1.38 Å and 2.26 Å resolution, respectively, providing the highest resolution structure for R2lox, as well as new insights into putative mechanisms regulating the function of the enzyme.
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| 2. |
- Diamanti, Riccardo, 1988-
(författare)
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Shining light on unknown function proteins
- 2022
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The study of protein structure and function is a fundamental step for understanding biological systems and for creating a solid ground for future drug development. Despite the development of novel ground-breaking biochemical and bioinformatical tools in the last decades, most proteins found in nature still have unknown assigned function. In this thesis, I present four publications aimed at protein structure and function determination. In these publications we present one method for high-throughput condition screening of membrane proteins for structural studies and three publications that aim at contributing to the understanding of proteins found in the pathogenic bacterium Mycobacterium tuberculosis, the deadliest bacterium worldwide, responsible for over 1 million deaths every year. Membrane proteins are very important targets for drug development. However, the study of membrane proteins is much more complex than fortheir soluble counterpart. In paper I, we developed a high-throughput method for detergent screening of membrane proteins that significantly reduces the time needed to find optimal conditions for structural studies. We developed amethod that allows for the screening of 192 protein-detergent conditions in 3 days. We used a TEV-folding reporter GFP-His tag vector to be able to measure the stability and amount of each sample after detergent solubilization of the cell lysate.In paper II, we characterized the Mycobacterium smegmatis respiratory supercomplex accessory proteins AscF and AscG. AscF and AscG arelocated in close proximity to the D and K proton pathways of Cytochrome c oxidase and might play a regulatory role in the respiratory supercomplex. Wes how how AscF and AscG do not bind nor metals nor nucleotides, but that they are essential for the normal growth of the M. smegmatis. We also show that both proteins are highly conserved in almost whole Mycobacteriales families, including M. tuberculosis. In paper III, we present two novel structures of the ferritin-like protein R2lox and further characterize the co-purified ligands in its lipid-binding pocket. We demonstrate how the two prevalent ligands that co-purify with the enzyme are fatty acids that host a C12 hydroxy-group and a double bond between the hydroxy group and the carboxylic group. The new structures from Sulfolobus acidocaldarius and Saccharopolyspora erythraea reveal new information about the enzyme’s possible regulatory mechanism and its possible interaction with partner proteins. In paper IV, we performed structural and functional studies of nicotinamide adenine dinucleotide (NAD+) synthase (NadE), which performs the last step of the de novo synthesis of NAD+: the amidation of nicotinic acidadenine dinucleotide (NaAD) into NAD+. We showed that some prokaryotes concert up to three NadE isoforms depending on external ammonium (NH3) availability. During high external concentrations of NH3, the ammonium-dependent NadE acts as primary catalyst of NAD+ synthesis. However, two glutamine-dependent homologues are capable of deamidation of glutamine when external ammonium availability is reduced. We show how the octameric glutamine-dependent NadE is the principal catalyst during nitrogen-fixing conditions, while the dimeric variant is the primary catalyst when NH3 availability is limited.
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| 3. |
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| 4. |
- Schenberger Santos, Adrian Richard, et al.
(författare)
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Kinetics and structural features of dimeric glutamine-dependent bacterial NAD(+) synthetases suggest evolutionary adaptation to available metabolites
- 2018
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 293:19, s. 7397-7407
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Tidskriftsartikel (refereegranskat)abstract
- NADH (NAD(+)) and its reduced form NADH serve as cofactors for a variety of oxidoreductases that participate in many metabolic pathways. NAD(+) also is used as substrate by ADP-ribosyl transferases and by sirtuins. NAD(+) biosynthesis is one of the most fundamental biochemical pathways in nature, and the ubiquitous NAD(+) synthetase (NadE) catalyzes the final step in this biosynthetic route. Two different classes of NadE have been described to date: dimeric single-domain ammonium-dependent NadE(NH3) and octameric glutamine-dependent NadE(Gln), and the presence of multiple NadE isoforms is relatively common in prokaryotes. Here, we identified a novel dimeric group of NadE(Gln) in bacteria. Substrate preferences and structural analyses suggested that dimeric NadE(Gln) enzymes may constitute evolutionary intermediates between dimeric NadE(NH3) and octameric NadE(Gln). The characterization of additional NadE isoforms in the diazotrophic bacterium Azospirillum brasilense along with the determination of intracellular glutamine levels in response to an ammonium shock led us to propose a model in which these different NadE isoforms became active accordingly to the availability of nitrogen. These data may explain the selective pressures that support the coexistence of multiple isoforms of NadE in some prokaryotes.
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| 5. |
- Sjöstrand, Dan, et al.
(författare)
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A rapid expression and purification condition screening protocol for membrane protein structural biology
- 2017
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Ingår i: Protein Science. - : Wiley. - 0961-8368 .- 1469-896X. ; 26:8, s. 1653-1666
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Tidskriftsartikel (refereegranskat)abstract
- Membrane proteins control a large number of vital biological processes and are often medically important-not least as drug targets. However, membrane proteins are generally more difficult to work with than their globular counterparts, and as a consequence comparatively few high-resolution structures are available. In any membrane protein structure project, a lot of effort is usually spent on obtaining a pure and stable protein preparation. The process commonly involves the expression of several constructs and homologs, followed by extraction in various detergents. This is normally a time-consuming and highly iterative process since only one or a few conditions can be tested at a time. In this article, we describe a rapid screening protocol in a 96-well format that largely mimics standard membrane protein purification procedures, but eliminates the ultracentrifugation and membrane preparation steps. Moreover, we show that the results are robustly translatable to large-scale production of detergent-solubilized protein for structural studies. We have applied this protocol to 60 proteins from an E. coli membrane protein library, in order to find the optimal expression, solubilization and purification conditions for each protein. With guidance from the obtained screening data, we have also performed successful large-scale purifications of several of the proteins. The protocol provides a rapid, low cost solution to one of the major bottlenecks in structural biology, making membrane protein structures attainable even for the small laboratory.
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