Towards crystallization of hydrophobic myelin glycoproteins: P0 and PASII/PMP22.
The preparation of a pure and homogeneous protein sample at proper concentration is a prerequisite for success when attempting their crystallization for structural determination. The detergents suitable for solubilization particularly of membrane proteins are not always the best for crystallization. Myelin of the peripheral nervous system of vertebrates is the example of a membrane for which neutral or "gentle" detergents are not even strong enough to solubilize its proteins. In contrast, sodium- or lithium-dodecyl sulfate is very effective. We solubilized myelin membrane in 2%(w/v) sodium dodecyl sulfate, followed by chromatographic purification of the hydrophobic myelin glycoproteins P0 and PASII/PMP22, and finally, we have exchanged the sodium dodecyl sulfate bound to protein for other neutral detergents using ceramic hydroxyapatite column. Theoretically, we should easily exchange sodium dodecyl sulfate for any neutral detergent, but for some of them, the solubility of myelin glycoproteins is low. To monitor the potential variability in the secondary structure of glycoproteins, we have used circular dichroism. Sodium dodecyl sulfate seems to be the appropriate detergent for the purpose of purification of very hydrophobic glycoproteins, since it can be easily exchanged for another neutral detergent.
Sedzik
Jan
J
Institute for Protein Research, Osaka University, Osaka 565-0871, Japan. Sedzik@swipnet.se
Uyemura
Keiichi
K
Tsukihara
Tomitake
T
D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003300:Copper; D003460:Crystallization / Q000379:methods*; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D057927:Hydrophobic and Hydrophilic Interactions; D007093:Imidazoles; D018993:Myelin P0 Protein / Q000737:chemistry*; D009185:Myelin Proteins / Q000737:chemistry*
D007093:Imidazoles; D018993:Myelin P0 Protein; D009185:Myelin Proteins; D003300:Copper; D017886:Durapatite; C005954:imidazoleacetic acid