A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins

Gan, Siok Wan; Vararattanavech, Ardcharaporn; Nordin, Nurhuda; Eshaghi, Said; Torres, Jaume

doi:10.1016/j.ab.2011.05.007

Cited by 15 publications

(21 citation statements)

References 47 publications

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“…Even in the presence of CATR, small amounts of AAC3 can aggregate into higher molecular mass species under particular conditions. Protein “laddering” has been observed with other membrane proteins (44, 45, 47, 48) and is consistent with the occurrence of less favorable solubilization conditions. This typically occurs when the detergent concentration is limiting but can occur at higher concentrations also with particular detergents ( e.g.…”

Section: Discussionsupporting

confidence: 73%

“…in digitonin or, on occasion, in 12M). In general, there is a good correlation between membrane protein profiles observed on blue native gels (taking into account correction factors) with those observed using other sizing techniques, suggesting that the real state of the protein in detergent is reported despite the presence of Coomassie dye (47, 48). Of note, minor AAC multimers were identified by analytical ultracentrifugation in preparations of bovine AAC1 in Triton X-100, where the protein was also found to be predominantly monomeric (30).…”

Section: Discussionmentioning

confidence: 74%

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Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

Crichton

Harding

Ruprecht

et al. 2013

Journal of Biological Chemistry

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Background: Mitochondrial carriers were thought to be dimeric based on their migration in blue native gels.Results: The high molecular mass species observed in blue native gels are composed of protein monomers, detergent, lipid, and Coomassie stain.Conclusion: The mitochondrial carriers are monomeric not dimeric.Significance: The apparent mass of small membrane proteins in blue native gels requires significant correction.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 74%

Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

Crichton

Harding

Ruprecht

et al. 2013

Journal of Biological Chemistry

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show abstract

“…3A). In common with other membrane proteins (Gan et al, 2011), the molecular weight of this band may not correspond to the molecular weight markers used, or to the AQPZ ladder (∼25 kDa for the monomer) possibly because of residual detergent bound to the protein. However, after dilution with 25 mM DPC, MERS-CoV E protein dissociated into lower molecular weight oligomers, from monomers to pentamers (Fig.…”

Section: Mers-cov E Protein Forms Pentamersmentioning

confidence: 97%

“…For SDS-NuPAGE, samples were run in 4-12% NuPAGE ® Bis-Tris gels (Invitrogen) with NuPAGE ® MES SDS running buffer and stained with SimplyBlue TM SafeStain (Invitrogen) according to the manufacturer's protocol. Blue-native PAGE (BN-PAGE) was performed as described previously (Gan et al, 2011). Lyophilized peptide was solubilized to 0.1 mM in a sample buffer containing 50 mM DHPC (Avanti Polar Lipids), 5, 25 and 50 mM DPC (Avanti Polar Lipids), or 21 mM LMPG (Anatrace).…”

Section: Gel Electrophoresismentioning

confidence: 99%

MERS coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels

Surya

Verdiá-Báguena

et al. 2015

Virus Research

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“…All these detergents are suitable for SE, since their density can be matched by addition of D 2 O. Samples should also be tested by sedimentation velocity (SV), which provides the minimum number of species present 34 . Also, unsuitable detergents will produce multiple species in SV, and multiple bands in PFO electrophoresis, indicative of multiple non-specific association.…”

Section: Discussionmentioning

confidence: 99%

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

Surya

Torres

2015

JoVE

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Analytical ultracentrifugation (AUC) can be used to study reversible interactions between macromolecules over a wide range of interaction strengths and under physiological conditions. This makes AUC a method of choice to quantitatively assess stoichiometry and thermodynamics of homo-and hetero-association that are transient and reversible in biochemical processes. In the modality of sedimentation equilibrium (SE), a balance between diffusion and sedimentation provides a profile as a function of radial distance that depends on a specific association model. Herein, a detailed SE protocol is described to determine the size and monomer-monomer association energy of a small membrane protein oligomer using an analytical ultracentrifuge. AUC-ES is label-free, only based on physical principles, and can be used on both water soluble and membrane proteins. An example is shown of the latter, the small hydrophobic (SH) protein in the human respiratory syncytial virus (hRSV), a 65-amino acid polypeptide with a single α-helical transmembrane (TM) domain that forms pentameric ion channels. NMR-based structural data shows that SH protein has two protonatable His residues in its transmembrane domain that are oriented facing the lumen of the channel. SE experiments have been designed to determine how pH affects association constant and the oligomeric size of SH protein.While the pentameric form was preserved in all cases, its association constant was reduced at low pH. These data are in agreement with a similar pH dependency observed for SH channel activity, consistent with a lumenal orientation of the two His residues in SH protein. The latter may experience electrostatic repulsion and reduced oligomer stability at low pH. In summary, this method is applicable whenever quantitative information on subtle protein-protein association changes in physiological conditions have to be measured. Video LinkThe video component of this article can be found at

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A cost-effective method for simultaneous homo-oligomeric size determination and monodispersity conditions for membrane proteins

Cited by 15 publications

References 47 publications

Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

Lipid, Detergent, and Coomassie Blue G-250 Affect the Migration of Small Membrane Proteins in Blue Native Gels

MERS coronavirus envelope protein has a single transmembrane domain that forms pentameric ion channels

Sedimentation Equilibrium of a Small Oligomer-forming Membrane Protein: Effect of Histidine Protonation on Pentameric Stability

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