Functional characterization and NMR spectroscopy on full-length Vpu from HIV-1 prepared by total chemical synthesis.
Vpu is an 81-residue integral membrane protein encoded in the HIV-1 genome that is of considerable interest because it plays important roles in the release of virus particles from infected cells and in the degradation of the cellular receptor. We report here the total chemical synthesis of full-length Vpu(1-81) as well as a site-specifically (15)N-labeled analogue, Vpu(2-81), using native chemical ligation methodologies and also report a structural and functional comparison of these constructs with recombinant protein obtained via bacterial expression. The structures of the synthetic and expressed polypeptides were similar in lipid micelles using solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in aligned hydrated lipid bilayers indicated that their overall topologies were also very comparable. Further, the channel activity of the synthetic protein was found to be analogous to that previously characterized for the recombinant protein. We have thus demonstrated that using solid phase peptide synthesis and chemical ligation it is feasible to obtain large quantities of a purified and homogeneous membrane protein in a structurally and functionally relevant form for future structural and characterization studies.
Gryphon Therapeutics, 600 Gateway Blvd., South San Francisco, California 94080, USA. gerd@gryphonRX.com
Gerd G
GG
Kochendoerfer
David H
DH
Jones
Sangwon
S
Lee
Myrta
M
Oblatt-Montal
Stanley J
SJ
Opella
Mauricio
M
Montal
D000595:Amino Acid Sequence; D015497:HIV-1 / Q000378:metabolism*; D054298:Human Immunodeficiency Virus Proteins; D007473:Ion Channels / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry; D054334:Viral Regulatory and Accessory Proteins / Q000138:chemical synthesis* / Q000737:chemistry*; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D007473:Ion Channels; D008051:Lipid Bilayers; D008823:Micelles; D011994:Recombinant Proteins; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins