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-leng...

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Bibliographic Details
Published in:Journal of the American Chemical Society 2004-03, Vol.126 (8), p.2439-2446
Main Authors: Kochendoerfer, Gerd G, Jones, David H, Lee, Sangwon, Oblatt-Montal, Myrta, Opella, Stanley J, Montal, Mauricio
Format: Article
Language:English
Subjects:
Amino Acid Sequence
Analytical, structural and metabolic biochemistry
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
General aspects, investigation methods
HIV-1 - metabolism
Human Immunodeficiency Virus Proteins
Ion Channels - chemistry
Lipid Bilayers - chemistry
Micelles
Molecular Sequence Data
Nuclear Magnetic Resonance, Biomolecular - methods
Proteins
Recombinant Proteins - chemistry
Viral Regulatory and Accessory Proteins - chemical synthesis
Viral Regulatory and Accessory Proteins - chemistry
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Summary: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 15N-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.
ISSN:0002-7863
1520-5126
DOI:10.1021/ja038985i