An amphipathic polypeptide derived from poly‐γ‐glutamic acid for the stabilization of membrane proteins

Abstract

Difficulties in the extraction of membrane proteins from cell membrane and their solubilization in native conformations have hindered their structural and biochemical analysis. To overcome these difficulties, an amphipathic polypeptide was synthesized by the conjugation of octyl and glucosyl groups to the carboxyl groups of poly-c-glutamic acid (PGA). This polymer, called amphipathic PGA (APG), self-assembles as mono-disperse oligomers consisted of 4-5 monomers. APG shows significantly low value of critical micelle concentration and stabilization activity toward membrane proteins. Most of the sodium dodecyl sulfate (SDS)-solubilized membrane proteins from Escherichia coli remain soluble state in the presence of APG even after the removal of SDS. In addition, APG stabilizes purified 7 transmembrane proteins such as bacteriorhodopsin and human endothelin receptor Type A (ET A ) in their active conformations. Furthermore, ET A in complex with APG is readily inserted into liposomes without disrupting the integrity of liposomes. These properties of APG can be applied to overcome the difficulties in the stabilization and reconstitution of membrane proteins.

Determination of the endothelin-1 recognition sites of endothelin receptor type A by the directed-degeneration method

“…We then reconstituted the proteins with APG, which was synthesized by the conjugation of alkyl and glucosyl groups to the carboxylic acid groups of poly-γ-glutamic acid and which facilitates the stabilization of P9-ET A in its active conformation 27 , 28 . Then, the ET-1 binding activities of the mutants compared to wild type P9-ET A were investigated (Fig.…”

“…3c ). To measure the relative binding activities of the purified proteins to ET-1, we applied 5 μM ET-1 to the immobilized proteins; this concentration is sufficient to cover the maximal binding capacity (B max ) of the receptors because it is about 100-fold higher than the apparent equilibrium dissociation constant (K D ) of the wild type P9-ET A complex with APG against ET-1 (60 nM) 27 . As shown in Fig.…”

“…The buffer of the eluted protein fraction was exchanged with buffer B using a PD-10 desalting column (GE, USA) to remove excess imidazole. The prepared proteins were stabilized in their active conformation with APG as previously described 27 , 28 . The proteins were stored with 10% glycerol at −80 °C until further use.…”

Development of Single‐Chain Antibodies Specific to Lysophosphatidic Acid Receptor 2

“…A recombinant human LPA 2 containing the N‐terminus P9 sequence (P9‐LPA 2 ) was purified as previously described with slight modification . In brief, the expression vector for LPA 2 (pP9*‐LPA2) was transformed into Rosetta(DE3) cells, and the transformed cells were grown at 37C until an OD 600 of 0.5–0.6.…”

“…The lipid suspension was sonicated in a 5 s pulse of 100 cycles using Sonosmasher (Sibata, Saitama, Japan) to produce small unilamellar vesicles (SUVs). Reconstitution of P9‐LPA 2 into SUVs was performed as described previously . Briefly, 100 μg of P9‐LPA 2 stabilized with APG–ODG was mixed with 1 mg of SUVs, and then incubated with shaking for 4 h at 4 °C.…”