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J. Biol. Chem., Vol. 279, Issue 17, 17483-17489, April 23, 2004

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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)^*

Christian F. W. Becker, Myrta Oblatt-Montal, Gerd G. Kochendoerfer¶, and Mauricio Montal||

From the Gryphon Therapeutics, South San Francisco, California 94080 and University of California, San Diego, La Jolla, California 92093-0366

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 T₄Vpu and T₅Vpu, exhibit the predicted mass as determined by MS analysis and fold with a high helical content as evidenced by CD. Both T₄Vpu and T₅Vpu, 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 T₄Vpu and 76 ± 5 pS for T₅Vpu 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.

Received for publication, December 3, 2003 , and in revised form, January 21, 2004.

^* This work was supported by Grants GM-49711 and GM-56538 from NIGMS, National Institutes of Health (to M. M.) and Grant GM62532 (to G. G. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–4.

¶ To whom correspondence may be addressed: Gryphon Therapeutics, 600 Gateway Blvd., South San Francisco, CA 94080. E-mail: gerd{at}gryphonRx.com. || To whom correspondence may be addressed: Section of Neurobiology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0366. E-mail: mmontal{at}ucsd.edu.

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