Chemical synthesis and single channel properties of tetrameric and pentameric TASPs (template-assembled synthetic proteins) derived from the transmembrane domain of HIV virus protein u (Vpu).
Vpu, an 81-residue membrane protein encoded by the genome of HIV-1, is involved in CD4 degradation and facilitates virion budding from infected cells. The latter activity requires an intact transmembrane (TM) domain; however, the mechanism remains unclear. Vpu forms ion channels, an activity linked to the TM domain and envisioned to arise by oligomerization. The precise number of Vpu monomers that structure the channel is not yet known. To address this issue, we have synthesized tetrameric and pentameric proteins consisting of a carrier template to which four or five peptides corresponding to the TM domain of Vpu are attached. Ketoxime-forming chemoselective ligation efficiently ligated four and five copies, respectively, of the linear transmembrane peptide that was solubilized by the addition of a cleavable polyethylene glycol-polyamide auxiliary to a template. Purified tetrameric and pentameric proteins, denoted as T(4)Vpu and T(5)Vpu, exhibit the predicted mass as determined by MS analysis and fold with a high helical content as evidenced by CD. Both T(4)Vpu and T(5)Vpu, after reconstitution in lipid bilayers, form discrete ion channels of distinct conductance and high propensity to be open. The most frequent openings have a single channel conductance of 42 +/- 5 pS for T(4)Vpu and 76 +/- 5 pS for T(5)Vpu in 0.5m KCl. These findings validate the notion that the channels formed by Vpu result from the self-assembly of monomers. We conclude that a five-helix bundle of the TM of Vpu may approximate the structural motif underlying the oligomeric state of the conductive channel.
Becker
Christian F W
CF
Gryphon Therapeutics, South San Francisco, California 94080, USA.
Oblatt-Montal
Myrta
M
Kochendoerfer
Gerd G
GG
Montal
Mauricio
M
D020816:Amino Acid Motifs; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D054298:Human Immunodeficiency Virus Proteins; D007473:Ion Channels / Q000737:chemistry; D007477:Ions / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry*; D013698:Templates, Genetic; D013997:Time Factors; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000235:genetics; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D007473:Ion Channels; D007477:Ions; D008051:Lipid Bilayers; D010455:Peptides; D011092:Polyethylene Glycols; D011506:Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins