Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase

Schmidt-Krey, Ingeborg; Lundqvist, Gerd; Morgenstern, Ralf; Hebert, Hans

doi:10.1006/jsbi.1998.4018

Cited by 31 publications

(21 citation statements)

References 39 publications

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“…Establishment of an overexpression system aimed at the production of active D1 protein is important in order to enable studies on cellular targetting and processing, as well as structure determination via either electron or X-ray crystallography (Schmidt-Krey et al 1998, Caffrey 2003. The overexpression of D1 protein represents a challenge because of several of its intrinsic properties: (1) the presence of an SeC residue in the catalytic center, (2) being an integral membrane protein, and (3) the requirement of a suitable native-like lipid environment essential for stability and catalytic activity upon purification (Mol et al 1988).…”

Section: Discussionmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in K m (2 µM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125 I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2-35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure-function analysis.

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Section: Discussionmentioning

confidence: 99%

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Kuiper

Klootwijk²,

Visser³

2005

Journal of Molecular Endocrinology

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“…Lipids were purchased from Avanti Polar Lipids as chloroform stocks. Lipid films were prepared by drying down lipid mixes under argon gas, as previously published, and storing them in glass vials at −30°C (35)(36)(37). To generate liposomes, the lipid films were hydrated with 100 mM NaCl/10 mM Mops at pH 7.4 and subjected to 5 rounds of rapid freeze-thaw cycles in liquid nitrogen and warm water.…”

Section: Methodsmentioning

confidence: 99%

Cellular distribution of copper to superoxide dismutase involves scaffolding by membranes

Pope

Feo

Unger

2013

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

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Significance What are the odds that a 1,000 Å 3 -sized metal-binding site will find its cargo parsing a volume of 30 trillion Å 3 by a random 3D diffusional process? Establishing a unique paradigm in the field of copper biology, we show that cells use a stunningly simple method to beat these odds by reducing the dimensionality of the problem through exploitation of cellular membranes as scaffolding components. We demonstrate that the copper delivery chaperone for the essential enzyme Cu/Zn superoxide dismutase 1 (SOD1) has the ability to bind to bilayers and that this previously unknown membrane binding interface is important for copper distribution to SOD1.

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“…36 The quality of the reconstituted crystals was evaluated by electron microscopy of negatively stained specimens. Excellent crystal preparations were selected for cryo-electron microscopy.…”

Section: Methodsmentioning

confidence: 99%

Structural Basis for Detoxification and Oxidative Stress Protection in Membranes

Holm

Bhakat

Jegerschöld

et al. 2006

Journal of Molecular Biology

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128

108

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Parameters for the Two-Dimensional Crystallization of the Membrane Protein Microsomal Glutathione Transferase

Cited by 31 publications

References 39 publications

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast

Cellular distribution of copper to superoxide dismutase involves scaffolding by membranes

Structural Basis for Detoxification and Oxidative Stress Protection in Membranes

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