The application of perfluorooctanoate to investigate trimerization of the human immunodeficiency virus-1 gp41 ectodomain by electrophoresis.
The transmembrane glycoprotein gp41 of human immunodeficiency virus has been proposed to form trimer-of-hairpin during virus-cell membrane fusion. To investigate its oligomerization propensity under soluble and membrane-mimic conditions, sodium salt of perfluorooctanoate (PFO) was applied. A recombinant gp41 ectodomain devoid of disulfide linkage was overexpressed in Escherichia coli and characterized by MS and circular dichroism spectropolarimetry in PFO solution in comparison to SDS. The helical content of this ectodomain in PFO is higher than that in SDS. Notably, PFO employed in PAGE clearly conduced to the formation of trimer under the optimized condition as visualized in the gel. In addition, the proteins expressed from the two mutants in the heptad repeat (HR) domains of gp41, I62P, and N126K, were also examined by the PFO-PAGE analysis for functional ramification of molecular organization. Remarkably, the I62P mutation completely abolished the gp41 trimer formation, whereas the N126K mutation resulted in a more stable trimer. The data suggested that PFO-PAGE analysis is appropriate for evaluating the effect of mutations on the trimerization of gp41 and other fusion proteins which may be implicated in the alteration of their fusogenicity.
Lin
Chi-Hui
CH
Institute of Chemistry, Academia Sinica, Taipei, Taiwan.
Chang
Chung-Chieh
CC
Cheng
Shu-Fang
SF
Chang
Ding-Kwo
DK
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001704:Biopolymers; D002210:Caprylates / Q000494:pharmacology*; D002942:Circular Dichroism; D003902:Detergents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D005466:Fluorocarbons / Q000494:pharmacology*; D015700:HIV Envelope Protein gp41 / Q000032:analysis / Q000737:chemistry* / Q000187:drug effects; D015497:HIV-1 / Q000737:chemistry*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D001704:Biopolymers; D002210:Caprylates; D003902:Detergents; D005466:Fluorocarbons; D015700:HIV Envelope Protein gp41; D011993:Recombinant Fusion Proteins; C514787:gp41 protein, Human immunodeficiency virus 1; D012967:Sodium Dodecyl Sulfate; C023036:perfluorooctanoic acid