Synthesis and characterization of the hydrophilic C-terminal domain of the human immunodeficiency virus type 1-encoded virus protein U (Vpu).
The HIV-1-specific virus protein U (Vpu) is an amphipathic membrane-associated phosphoprotein that probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. Because of an as yet undefined cytotoxic effect and the low concentration of Vpu in host cells, it has not been possible to obtain sufficient quantities of this protein for biochemical and structural investigations. We describe the synthesis, in two forms, of 50-residue peptides representing the polar cytoplasmic domain of Vpu: pVpu, comprising the wild-type Vpu sequence of HIV-1, strain HTLVIIIB, from position Ile32 to Leu81, and a mutant, pVpum2/6, with replacement of Ser52-56 with Asn. This mutation affects the two phosphorylation sites of Vpu for casein kinase-2 (CK-2), the enzyme that phosphorylates Vpu in HIV-1-infected cells. The peptides were purified to homogeneity and characterized by N-terminal sequencing, mass spectrometry and SDS-PAGE. Furthermore, both peptides were immunologically characterized by Western blot and ELISA using Vpu-specific monoclonal and polyclonal antibodies. Recombinant human CK-2 caused in vitro phosphorylation of pVpu, but had no effect on the mutant pVpum2/6. Investigation of the peptides by circular dichroism showed that addition of trifluoroethanol stabilized the alpha-helical secondary structure. Preliminary 1H NMR spectroscopic data indicated that, in the presence of trifluoroethanol, both peptides in solution have stable secondary structures with considerable alpha-helical content.
Henklein
P
P
Institut für Pharmakologie und Toxikologie, Medizinische Fakultät (Charité), Humboldt-Universität zu Berlin, Germany.
Schubert
U
U
Kunert
O
O
Klabunde
S
S
Wray
V
V
Klöppel
K D
KD
Kiess
M
M
Portsmann
T
T
Schomburg
D
D
D000163:Acquired Immunodeficiency Syndrome; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002942:Circular Dichroism; D015497:HIV-1 / Q000737:chemistry*; D054298:Human Immunodeficiency Virus Proteins; D007124:Immunoenzyme Techniques; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000737:chemistry* / Q000378:metabolism; D000086082:Viroporin Proteins
D054298:Human Immunodeficiency Virus Proteins; D010446:Peptide Fragments; D010770:Phosphotransferases; D054334:Viral Regulatory and Accessory Proteins; C514859:vpu protein, Human immunodeficiency virus 1; D000086082:Viroporin Proteins