Dataset Information

Dodecyl-?-melibioside Detergent Micelles as a Medium for Membrane Proteins.

ABSTRACT: There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-?-melibioside (?-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-?(1?6)-d-glucose. Light scattering showed the ?-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-?-maltoside (?-DDM). ?-DDMB stabilized diacylglycerol kinase (DAGK) against thermal inactivation. Moreover, activity assays conducted using aliquots of DAGK purified into ?-DDMB yielded activities that were 40% higher than those of DAGK purified into ?-DDM. ?-DDMB yielded similar or better TROSY-HSQC NMR spectra for two single-pass membrane proteins and the tetraspan membrane protein peripheral myelin protein 22. ?-DDMB appears be a useful addition to the toolbox of non-ionic detergents available for membrane protein research.

SUBMITTER: Hutchison JM

PROVIDER: S-EPMC5685800 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Dodecyl-β-melibioside Detergent Micelles as a Medium for Membrane Proteins.

Hutchison James M JM Lu Zhenwei Z Li Geoffrey C GC Travis Benjamin B Mittal Ritesh R Deatherage Catherine L CL Sanders Charles R CR

Biochemistry 20171009 41

There remains a need for new non-ionic detergents that are suitable for use in biochemical and biophysical studies of membrane proteins. Here we explore the properties of n-dodecyl-β-melibioside (β-DDMB) micelles as a medium for membrane proteins. Melibiose is d-galactose-α(1→6)-d-glucose. Light scattering showed the β-DDMB micelle to be roughly 30 kDa smaller than micelles formed by the commonly used n-dodecyl-β-maltoside (β-DDM). β-DDMB stabilized diacylglycerol kinase (DAGK) against thermal i ...[more]

PMID: 28980804

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Project description:Membrane proteins are largely dependent for their function on the phospholipids present in their immediate environment, and when they are solubilized by detergent for further study, residual phospholipids are critical, too. Here, brominated phosphatidylcholine, a phospholipid which behaves as an unsaturated phosphatidylcholine, was used to reveal the kinetics of phospholipid exchange or transfer from detergent mixed micelles to the environment of a detergent-solubilized membrane protein, the paradigmatic P-type ATPase SERCA1a, in which Trp residues can experience fluorescence quenching by bromine atoms present on phospholipid alkyl chains in their immediate environment. Using dodecylmaltoside as the detergent, exchange of (brominated) phospholipid was found to be much slower than exchange of detergent under the same conditions, and also much slower than membrane solubilization, the latter being evidenced by light scattering changes. The kinetics of this exchange was strongly dependent on temperature. It was also dependent on the total concentration of the mixed micelles, revealing the major role for such exchange of the collision of detergent micelles with the detergent-solubilized protein. Back-transfer of the brominated phospholipid from the solubilized protein to the detergent micelle was much faster if lipid-free DDM micelles instead of mixed micelles were added for triggering dissociation of brominated phosphatidylcholine from the solubilized protein, or in the additional presence of C12E8 detergent during exchange, also emphasizing the role of the chemical nature of the micelle/protein interface. This protocol using brominated lipids appears to be valuable for revealing the possibly slow kinetics of phospholipid transfer to or from detergent-solubilized membrane proteins. Independently, continuous recording of the activity of the protein can also be used in some cases to correlate changes in activity with the exchange of a specific phospholipid, as shown here by using the Drs2p/Cdc50p complex, a lipid flippase with specific binding sites for lipids.

Dataset Information

Dodecyl-?-melibioside Detergent Micelles as a Medium for Membrane Proteins.

Publications

Dodecyl-β-melibioside Detergent Micelles as a Medium for Membrane Proteins.

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