Expression of human proteins at the Southeast Collaboratory for Structural Genomics

Mayer, Michael R.; Dailey, Tamara A.; Baucom, Clay M.; Supernak, Jill L.; Grady, Michael C.; Hawk, Harris; Dailey, Harry A.

doi:10.1023/b:jsfg.0000029202.77832.34

Cited by 15 publications

(11 citation statements)

References 26 publications

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“…Wild-type and F110A variant ferrochelatases were expressed and purified as previously described 35,36. Prior to affinity purification using Talon™ (Clontech, Palo Alto, California) metal affinity resin, approximately 10 µmole porphyrin, either deuteroporphyrin IX or protoporphyrin IX (Frontier Scientific, Logan, Utah) and metal salt (HgCl 2 , CdCl 2 , MnCl 2 or NiCl 2 ) were added to crude extracts.…”

Section: Methodsmentioning

confidence: 99%

Product Release Rather than Chelation Determines Metal Specificity for Ferrochelatase

Medlock

Carter

Dailey

et al. 2009

Journal of Molecular Biology

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SUMMARYFerrochelatase (protoheme ferrolyase, E.C. 4.99.1.1) is the terminal enzyme in heme biosynthesis and catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX (heme). Within the past two years x-ray crystallographic data obtained with human ferrochelatase have clearly shown that significant structural changes occur during catalysis that are predicted to facilitate metal insertion and product release. One unanswered question about ferrochelatase involves defining the mechanism whereby some metals, such as divalent Fe, Co, Ni and Zn, can be used by the enzyme in vitro to produce the corresponding metalloporphyrins while other metals, such as divalent Mn, Hg, Cd or Pb, are inhibitors of the enzyme. Through the use of high resolution x-ray crystallography along with characterization of metal species via their anomalous diffraction, the identity and position of Hg, Cd, Ni, or Mn in the center of enzyme-bound porphyrin macrocycle was determined. When Pb, Hg, Cd or Ni was present in the macrocycle the conserved π helix was in the extended, partially unwound "product release" state. Interestingly, in the structure of ferrochelatase with Mn-porphyrin bound the π helix is not extended or unwound and is in the "substrate-bound" conformation. These findings show that at least in the cases of Mn, Pb, Cd and Hg, metal "inhibition" of ferrochelatase is not due to the inability of the enzyme to insert the metal into the macrocycle or by binding to a second metal binding site as has been previously proposed. Rather, inhibition occurs after metal insertion and results from poor or diminished product release. Possible explanations for the lack of product release are proposed herein.

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Section: Methodsmentioning

confidence: 99%

Product Release Rather than Chelation Determines Metal Specificity for Ferrochelatase

Medlock

Carter

Dailey

et al. 2009

Journal of Molecular Biology

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“…Mutagenesis, expression, and purification of human ferrochelatase were performed as described (8,26,37). Concentration buffer for variants consisted of 0.045 M Tris-Mops (pH 8.1), 0.09 M potassium chloride, 0.9% sodium cholate, 0.225 M imidazole, and 10% glycerol.…”

Section: Methodsmentioning

confidence: 99%

Substrate interactions with human ferrochelatase

Medlock

Swartz

Dailey

et al. 2007

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

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Ferrochelatase, the terminal enzyme in heme biosynthesis, catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme IX. Human ferrochelatase is a homodimeric, inner mitochondrial membrane-associated enzyme that possesses an essential [2Fe-2S] cluster. In this work, we report the crystal structure of human ferrochelatase with the substrate protoporphyrin IX bound as well as a higher resolution structure of the R115L variant without bound substrate. The data presented reveal that the porphyrin substrate is bound deep within an enclosed pocket. When compared with the location of N-methylmesoporphyrin in the Bacillus subtilis ferrochelatase, the porphyrin is rotated by Ϸ100°and is buried an additional 4.5 Å deeper within the active site. The propionate groups of the substrate do not protrude into solvent and are bound in a manner similar to what has been observed in uroporphyrinogen decarboxylase. Furthermore, in the substrate-bound form, the jaws of the active site mouth are closed so that the porphyrin substrate is completely engulfed in the pocket. These data provide insights that will aid in the determination of the mechanism for ferrochelatase.heme biosynthesis ͉ protoporphyrin IX ͉ x-ray crystallography ͉ metal insertion

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“…However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise [1]. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes.…”

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confidence: 99%

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et al. 2005

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Background: Protein expression in E. coli is the most commonly used system to produce protein for structural studies, because it is fast and inexpensive and can produce large quantity of proteins. However, when proteins from other species such as mammalian are produced in this system, problems of protein expression and solubility arise [1]. Structural genomics project are currently investigating proteomics pipelines that would produce sufficient quantities of recombinant proteins for structural studies of protein complexes. To investigate how the E. coli protein expression system could be used for this purpose, we purified apoptotic binary protein complexes formed between members of the Caspase Associated Recruitment Domain (CARD) family.

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Expression of human proteins at the Southeast Collaboratory for Structural Genomics

Cited by 15 publications

References 26 publications

Product Release Rather than Chelation Determines Metal Specificity for Ferrochelatase

Product Release Rather than Chelation Determines Metal Specificity for Ferrochelatase

Substrate interactions with human ferrochelatase

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