A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research

Urbani, Andrea; Warne, Tony

doi:10.1016/j.ab.2004.09.040

Cited by 69 publications

(62 citation statements)

References 23 publications

(31 reference statements)

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“…For data analysis, protein was taken to have a refractive index increment (dn/dc) of 0.186 ml/g and DDM of 0.143 ml/g (26). The ratio of DDM to protein was determined by a colorimetric assay of the DDM maltoside (25) as 219 Ϯ 63 DDM per VcSiaQM and 235 Ϯ 93 DDM per HiSiaQM. These ratios were used to calculate a composite dn/dc of 0.159 ml/g for HiSiaQM-DDM and VcSiaQM-DDM complexes (27).…”

Section: Methodsmentioning

confidence: 99%

“…5 mM EDTA was added to the elution fractions. DDM concentrations were determined using the methods of Urbani and Warne (25).…”

Section: Methodsmentioning

confidence: 99%

“…6C) (25). For VcSiaQM, the DDM assay revealed that there were 219 Ϯ 63 DDM molecules per 1:1 complex of VcSiaQM, giving a detergent mass component of 112 Ϯ 32 kDa.…”

Section: Failure Of Separately Expressed Membrane Domains To Form a Fmentioning

confidence: 99%

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The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Mulligan

Leech

Kelly

et al. 2012

Journal of Biological Chemistry

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Background: Vibrio cholerae has a need for sialic acid transport and catabolism for colonization. Results: The VC1777-1779 genes encode a functional tripartite ATP-independent periplasmic (TRAP) transporter for sialic acid in which the membrane subunits form a tight complex. Conclusion: Vibrio cholerae encodes a functional sialic acid TRAP transporter. Significance: This study reveals the route of sialic acid acquisition by an important human pathogen.

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Section: Methodsmentioning

confidence: 99%

“…5 mM EDTA was added to the elution fractions. DDM concentrations were determined using the methods of Urbani and Warne (25).…”

Section: Methodsmentioning

confidence: 99%

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The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

Mulligan

Leech

Kelly

et al. 2012

Journal of Biological Chemistry

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“…0.024% DDM in sizeexclusion chromatography buffer), after concentration for crystallization the detergent concentration could be as high as 0.5-3%. It is useful to check the detergent concentration [see Strop & Brunger (2005) for a list of methods for this; the authors find the technique described in Urbani & Warne (2005) to be useful] and test different amounts of detergent if there are crystal-stability issues.…”

Section: Finding a Suitable Mother Liquor/soaking Solution To Stabilimentioning

confidence: 99%

An overview of heavy-atom derivatization of protein crystals

Pike

Garman

Krojer

et al. 2016

Acta Crystallogr D Struct Biol

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Heavy-atom derivatization is one of the oldest techniques for obtaining phase information for protein crystals and, although it is no longer the first choice, it remains a useful technique for obtaining phases for unknown structures and for low-resolution data sets. It is also valuable for confirming the chain trace in lowresolution electron-density maps. This overview provides a summary of the technique and is aimed at first-time users of the method. It includes guidelines on when to use it, which heavy atoms are most likely to work, how to prepare heavy-atom solutions, how to derivatize crystals and how to determine whether a crystal is in fact a derivative.

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“…All of the detergents could extract the TP protein from the cell membrane and maintain the receptor ligand binding activity. The detergent, OG, which had successfully been used in the purification of many membrane proteins for structural studies (28)(29)(30), was selected for the TP purification first. Figure 4 shows the different purity levels of the TP protein purified after OG extraction and removal of the insoluble membrane proteins using ultracentrifugation ( Figure …”

Section: Purification Of the Tp Receptormentioning

confidence: 99%

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies

Ruan

Cervantes

2008

Biochemistry

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Human thromboxane A2 receptor (TP), a G protein-coupled receptor (GPCR), is one of the most promising targets for developing the next generation of anti-thrombosis and hypertension drugs. However, obtaining a sufficient amount of the full sized and active membrane protein has been the major obstacle for structural elucidation that reveals the molecular mechanisms of the receptor activation and drug designs. Here we report an approach for the simple, quick, and high-yield preparation of the purified and active full sized TP in an amount suitable for structural studies. Glycosylated human TP was highly expressed in Sf-9 cells using an optimized baculovirus (BV) expression system. The active receptor was extracted and solubilized by several different detergents for comparison, and was finally purified to a single band at a nearly perfect ratio of 1:0.9 ± 0.05 (ligand:receptor molecule) in ligand binding using a Ni-column with a relatively low yield. However, a high-yield purification (milligram quantity) of the TP protein, from a modulate scale of transfected Sf-9 cell culture, has been achieved by quick and simple purification steps, which include DNAdigestion, DM detergent-extraction, centrifugation and FPLC-purification. The purity and quantity of the purified TP, using the high-yield approach, was suitable for protein structural studies as evidenced by SDS-PAGE, Western blot analyses, ligand binding assays, and a feasibility test using high-resolution 1D and 2D 1 H NMR spectroscopic analyses. These studies provide a basis for the high-yield expression and purification of the GPCR for the structural and functional characterization using biophysics approaches.

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A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research

Cited by 69 publications

References 23 publications

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

An overview of heavy-atom derivatization of protein crystals

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies

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A colorimetric determination for glycosidic and bile salt-based detergents: applications in membrane protein research

Cited by 69 publications

References 23 publications

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

The Membrane Proteins SiaQ and SiaM Form an Essential Stoichiometric Complex in the Sialic Acid Tripartite ATP-independent Periplasmic (TRAP) Transporter SiaPQM (VC1777–1779) from Vibrio cholerae

An overview of heavy-atom derivatization of protein crystals

A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A2 Receptor in Full Size for Structural Studies

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A Simple, Quick, and High-Yield Preparation of the Human Thromboxane A₂ Receptor in Full Size for Structural Studies