A flexible and economical medium-throughput strategy for protein production and crystallization

Moreland, Nicole J.; Ashton, Rachael; Baker, Heather M.; Ivanović, Ivan; Patterson, Sean I.; Arcus, Vickery L.; Baker, Edward N.; Lott, J.S.

doi:10.1107/s0907444905023590

Cited by 74 publications

(63 citation statements)

References 27 publications

(27 reference statements)

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“…Rv1738 was readily expressed in E. coli as a soluble, His 6 -tagged protein at expression levels of 10 mg/L. After purification, crystallization trials were undertaken using a Cartesian nanoliter dispensing robot, with a 480-component screen (14). No crystals were obtained, either with or without the His 6 -tag.…”

Section: Resultsmentioning

confidence: 99%

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Bunker

Mandal

Bashiri

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Protein 3D structure can be a powerful predictor of function, but it often faces a critical roadblock at the crystallization step. Rv1738, a protein from Mycobacterium tuberculosis that is strongly implicated in the onset of nonreplicating persistence, and thereby latent tuberculosis, resisted extensive attempts at crystallization. Chemical synthesis of the L-and D-enantiomeric forms of Rv1738 enabled facile crystallization of the D/L-racemic mixture. The structure was solved by an ab initio approach that took advantage of the quantized phases characteristic of diffraction by centrosymmetric crystals. The structure, containing L-and D-dimers in a centrosymmetric space group, revealed unexpected homology with bacterial hibernation-promoting factors that bind to ribosomes and suppress translation. This suggests that the functional role of Rv1738 is to contribute to the shutdown of ribosomal protein synthesis during the onset of nonreplicating persistence of M. tuberculosis.

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Section: Resultsmentioning

confidence: 99%

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Bunker

Mandal

Bashiri

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…The boundary between the N-terminal DNA binding domain and the C-terminal ligand binding domain (C-PhnF) was located by alignment with the gene sequence of PhnF from Escherichia coli (25). C-phnF and full-length phnF were cloned into the Gateway system using the nested two-stage PCR protocol described previously (26,27) with the following primers: full-length phnF forward primer 5=-GGCAGCGGCG CGGTGACAGCGGGCGCG-3=, C-phnF forward primer 5=-GGCAGCG-GCGCGATCAGACAACCCCTCGGCATG-3, and full-length and C-phnF reverse primer 5=-GAAAGCTGGGTGTCACGAAACGATTGCGG-3=. Clones were verified by sequencing and transferred into the pDEST17 expression vector to produce a His-tagged protein (C-PhnF) or pD-EST566 to produce a His-tagged maltose-binding protein (MBP) fusion protein (full-length PhnF).…”

Section: Methodsmentioning

confidence: 99%

“…Initial crystallization conditions were obtained using a Honeybee (Cartesian Dispensing Systems) nanoliter robotic system in a sitting-drop format with 100 nl of protein solution mixed with 100 nl well solution in Intelliplates (Art Robbins Instruments) and a set of in-house screens covering 480 conditions (27).…”

Section: Methodsmentioning

confidence: 99%

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

et al. 2014

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dBacterial uptake of phosphate is usually accomplished via high-affinity transporters that are commonly regulated by two-component systems, which are activated when the concentration of phosphate is low. Mycobacterium smegmatis possesses two such transporters, the widely distributed PstSCAB system and PhnDCE, a transporter that in other bacteria mediates the uptake of alternative phosphorus sources. We previously reported that the transcriptional regulator PhnF controls the production of the Phn system, acting as a repressor under high-phosphate conditions. Here we show that the phnDCE genes are common among environmental mycobacteria, where they are often associated with phnF-like genes. In contrast, pathogenic mycobacteria were not found to encode Phn-like systems but instead were found to possess multiple copies of the pst genes. A detailed biochemical analysis of PhnF binding to its identified binding sites in the phnD-phnF intergenic region of M. smegmatis has allowed us to propose a quantitative model for repressor binding, which shows that a PhnF dimer binds independently to each site. We present the crystal structure of M. smegmatis PhnF at 1.8-Å resolution, showing a homodimer with a helix-turn-helix N-terminal domain and a C-terminal domain with a UbiC transcription regulator-associated fold. The C-terminal domain crystallized with a bound sulfate ion instead of the so far unidentified physiological ligand, allowing the identification of residues involved in effector binding. Comparison of the positioning of the DNA binding domains in PhnF with that in homologous proteins suggests that its DNA binding activity is regulated via a conformational change in the linker region, triggering a movement of the N-terminal domains.

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“…The cloning was conducted using the Gateway cloning system (21). The amplified PCR product was used to produce entry clones by performing a BP reaction.…”

Section: Pcr Amplification and Cloning-mentioning

confidence: 99%

“…Positive entry clones were selected on LB agar medium supplemented with 50 g/ml kanamycin and were then verified using BsrGI digestion and sequencing. The resulting entry clones were used to clone the full-length construct into pDEST17 (21) and pDESTsmg (22) vectors using an LR reaction. The expression construct for pDEST17 was selected on LB agar plates containing 100 g/ml ampicillin.…”

Section: Pcr Amplification and Cloning-mentioning

confidence: 99%

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

Bashiri

Rehan

Sreebhavan

et al. 2016

Journal of Biological Chemistry

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Cofactor F 420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis, the causative agent of tuberculosis. A ␥-glutamyl ligase catalyzes the final steps of the F 420 biosynthesis pathway by successive additions of L-glutamate residues to F 420 -0, producing a poly-␥-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F 420 -2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a two-domain protein and produces F 420 with predominantly 5-7 L-glutamate residues in the poly-␥-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated ␥-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiple L-glutamate residues to F 420 -0 in vitro to produce F 420 -5 after 24 h; communication between the two domains is critical for full ␥-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F 420 -0-, and FMN-bound states, displaying distinct sites for F 420 -0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N-and C-terminal domains in catalysis.The cofactor F 420 is a flavin derivative that is sporadically distributed among microorganisms, mainly archaea and actinobacteria (including mycobacteria). F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2-8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11). At the biochemical level, cofactor F 420 functions as a hydride transfer agent in oxidoreductive reactions with a lower redox potential than that of NAD(P) ϩ (12). The biosynthesis pathway of cofactor F 420 has been investigated in both archaeal and mycobacterial species. In the current view of the proposed pathway, the first intermediate with the complete chromophore (7, ) is produced by FO synthase (FbiC in mycobacteria (13) and CofGH in archaea (14)). A transferase enzyme (FbiA in mycobacteria (15) and CofD in archaea (16)) subsequently catalyzes the addition of a 2-phospho-L-lactate moiety to FO to produce F 420 -0 (F 420 with no poly-␥-glutamate tail). The final step of the pathway is performed by a ␥-glutamyl ligase (FbiB in mycobacteria (15) and CofE in archaea (17)) that catalyzes successive additions of L-glutamate residues to F 420 -0 (Fig. 1A).The length of the poly-␥-glutamate tail varies between archaeal and mycobacterial species; in archaea, two L-glutamate residues are seen (18), whereas in mycobacteria, up to nine residues are present (3, 19). There exists an intriguing difference between the ...

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A flexible and economical medium-throughput strategy for protein production and crystallization

Cited by 74 publications

References 27 publications

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

A functional role of Rv1738 in Mycobacterium tuberculosis persistence suggested by racemic protein crystallography

Crystal Structure of PhnF, a GntR-Family Transcriptional Regulator of Phosphate Transport in Mycobacterium smegmatis

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

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