Chemical engineering of Mycobacterium tuberculosis dodecin hybrids

Vinzenz, Xenia; Große, Wolfgang; Linne, Uwe; Meissner, Britta; Essen, Lars‐Oliver

doi:10.1039/c1cc12929e

Cited by 7 publications

(23 citation statements)

References 21 publications

(21 reference statements)

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“…If such an electron transfer occurs, the spectrum should show the absorption characteristics of a cationic tryptophan radical, expected around 560 nm (38,39) and of an anionic flavosemiquinone around 510 nm (40 -42). For RfbP from chicken egg white, an electron-transferred state consisting of a cationic tryptophan radical and a flavosemiquinone has already been identified which shows spectral characteristics as observed for dodecin (7,17). However, a remarkable difference between the reported data on chicken RfbP is the positive signal around 500 nm, which is not synchronized with the positive signal at longer wavelengths, and decays more slowly.…”

Section: Resultscontrasting

confidence: 43%

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Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Staudt

Oesterhelt

Grininger

et al. 2012

Journal of Biological Chemistry

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Background: Dodecin prevents riboflavin from autodegradation and exerting photo-induced cellular stress. Results: Ultrafast depopulation of excited states and ground state recovery is observed by transient spectroscopy. Conclusion: Ultrafast electron transfer in combination with proton transfer is responsible for deactivation of flavin excited states. Significance: A comprehensive study of parameters in the binding pocket affecting the riboflavin quenching mechanism is given.

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Section: Resultscontrasting

confidence: 43%

“…Structures revealed a dodecameric fold, providing six identical binding pockets, which each enables binding of two antiparallel arranged flavins between two tryptophan residues building an aromatic tetrade arrangement ( Fig. 1A) (1)(2)(3)(4)(5)(6)(7).…”

mentioning

confidence: 99%

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Staudt

Oesterhelt

Grininger

et al. 2012

Journal of Biological Chemistry

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“…There are fewer complexes with an odd number of subunits than there are complexes with an even number of subunits. Examples shown are dodecin (PDB ID 2yiz [60]), ferritin (PDB ID 1aew [61]), and Paramecium bursaria Chlorella virus type 1 (PDB ID 1m4x [62]).…”

Section: Trendsmentioning

confidence: 99%

Trendspotting in the Protein Data Bank

et al. 2013

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The Protein Data Bank (PDB) was established in 1971 as a repository for the three dimensional structures of biological macromolecules. Since then, more than 85,000 biological macromolecule structures have been determined and made available in the PDB archive. Through analysis of the corpus of data, it is possible to identify trends that can be used to inform us about the future of structural biology and to plan the best ways to improve the management of the ever-growing amount of PDB data.

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“…[1] Unique to dodecins is the binding mode of the flavins, where two flavins are bound between two tryptophans forming an aromatic tetrad in the binding pocket. [1,13,51] The crystal structure of HsDod revealed that the dodecin monomers have a β1α1β2β3 topology resulting in single αhelix partly enwrapped by a three stranded antiparallel β-sheets. [1] So far, all studied dodecins share this structure (see Figure 5).…”

Section: The Dodecin Protein Familymentioning

confidence: 99%

“…HhDod: The dodecin of H. halophila (PDB ID: 2VXA) [7] . MtDod: The dodecin of M. tuberculosis (PDB ID: 2YIZ) [51] . ScDod: The dodecin of Streptomyces coelicolor (PDB ID: 6R1E) [53] .…”

Section: The Dodecin Protein Familymentioning

confidence: 99%

The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications

Bourdeaux¹

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Biological Function of Bacterial Dodecins In this thesis, the dodecins of Mycobacterium tuberculosis (MtDod), Streptomyces coelicolor (ScDod) and Streptomyces davaonensis (SdDod) were studied. Kinetic measurements of the flavin binding of MtDod revealed that the dodecin binding pocket is filled in two distinct steps, for which a kinetic model then was established and verified by experimental data. The analysis with the two-step model showed that the unique binding pocket of dodecins allows them to bind excessive amounts of flavins, while at low flavin concentrations, flavin is released and only weakly bound. This function of flavin buffering prevents accumulation of free oxidised flavins and therefore helps to keep the redox balance of the cell and prevents potential cell damage caused by excessive free flavins. To further gain insights into the role of bacterial dodecins, the effect of knocking out the dodecin encoding gene in S. davaonensis was analysed. The knockout strain showed increased concentrations of various stress related metabolites, indicating that without dodecin the cellular balance is disrupted, which supports the role of dodecins as a flavin homeostasis factor. With a self-designed affinity measurement method based on the temperature dependent dissociation of the dodecin:flavin complex, which allowed parallel screening of multiple conditions, it was shown that MtDod, ScDod and SdDod have much higher affinities towards FMN and FAD under acidic conditions. Under these conditions, the three dodecins might function as a FMN storage. M. tuberculosis encounters multiple acidic environments during its infection cycle of humans and can adopt a state of dormancy. During recovery from the dormant state, a flavin storage might be beneficial. For some Streptomyces species it was reported that the formed spores are slightly acidic and therefore ScDod and SdDod could function as flavin storages for the spores. Further details on the flavin binding mechanism of MtDod were revealed by a mutagenesis study, identifying the importance of a histidine residue at the fourth position of the protein sequence for flavin binding, but contrary to expectations, this residue seems only to be partly involved in the pH related affinity shift. The data, reported in this thesis, demonstrates that bacterial dodecins likely function as flavin homeostasis factors, which allow overall higher flavin pools in the cell without disrupting the cellular balance. Further, the reported acid-dependent increase in binding affinity suggests that under certain conditions bacterial dodecins can also function as a flavin storage system. Application of the Dodecin of M. tuberculosis In this thesis, the stability of MtDod, ScDod SdDod and HsDod was analysed to find a suitable dodecin for the use as a carrier/scaffold. Therefore, a method to easily measure the stability of dodecins was designed, which measures the ability of the dodecamer to rebind flavins after a heating phase with stepwise increasing temperatures. Using this assay and testing the stability against detergents by SDS PAGE, showed that the dodecamer of MtDod possesses an excellent stability against a vast array of conditions, like temperatures above 95 °C, low pH and about 2% SDS. By solving the crystal structure of ScDod and SdDod, the latter forming a less stable dodecamer, combined with a mutagenesis study, the importance of a specific salt bridge for dodecamer stability was revealed and might be helpful to find further highly stable dodecins. In addition to the intrinsic high stability of the MtDod dodecamer, also the robustness of the fold was tested by creating diverse MtDod fusion constructs and producing them in Escherichia coli. Here it was shown that MtDod easily tolerates the attachment of proteins up to 4-times of its own size and that both termini can be modified without affecting the dodecamer noticeably. Further, it was shown that MtDod and many MtDod fusion constructs could be purified in high yields via a protocol based on the removal of E. coli proteins through heat denaturation and subsequent centrifugation. In a case study, by fusing diverse antigens from mostly human proteins to MtDod and using these constructs to produce antibodies in rabbits, it was demonstrated that MtDod is immunogenic and presents the attached antigens to the immune system. The here reported properties of MtDod and to a lesser degree of other bacterial dodecins, show that bacterial dodecins are a valuable addition to the pool of scaffold and carrier proteins and have great potential as antigen carriers.

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Chemical engineering of Mycobacterium tuberculosis dodecin hybrids

Cited by 7 publications

References 21 publications

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin

Trendspotting in the Protein Data Bank

The dodecin of Mycobacterium tuberculosis : biological function and biotechnical applications

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