Sequence-specific resonance assignments of the 1H-NMR spectra and structural characterization in solution of the HIV-1 transframe protein p6.
The frameshift protein p6* encoded directly upstream of the protease in the human immunodeficiency virus type 1 (HIV-1) pol reading frame is thought to be a natural inhibitor of protease activation and to play a role in the polyprotein processing of Gag and Gag-Pol precursors. To allow structural characterization of the p6* transframe protein, the p6* coding region was cloned into the vector pGEX-KG and expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST) under the control of the tac promoter. Thrombin cleavage of the construct resulted in a 70-amino-acid polypeptide which is extended by two additional residues at the N-terminus compared to the natural p6* sequence. The native purification procedure including an affinity and a size-exclusion chromatography step yielded sufficient amounts of highly pure protein suitable for NMR spectroscopy. Fluorescence, circular dichroism and 1H-NMR spectroscopy were applied to characterize the structure of protein. Two-dimensional NMR spectra provided essentially complete sequence-specific resonance assignments at pH 5.9. Although there is evidence for a helix-forming tendency in the N-terminus of the protein, the experiments indicate that p6* has no overall stable secondary or tertiary structure with the single tryptophan exposed in aqueous solution. However, the results reported herein open the way to characterize further the interaction of p6* with the HIV-1 protease in structural and functional in vitro studies.
Beissinger
M
M
Lehrstuhl für Biopolymere, Universität Bayreuth, Germany.
Paulus
C
C
Bayer
P
P
Wolf
H
H
Rösch
P
P
Wagner
R
R
D000595:Amino Acid Sequence; D001483:Base Sequence; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D015685:Fusion Proteins, gag-pol / Q000378:metabolism; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics*; D016333:HIV Protease / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D015394:Molecular Structure; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D012996:Solutions; D013053:Spectrophotometry; D054301:gag Gene Products, Human Immunodeficiency Virus
D004279:DNA, Viral; D015685:Fusion Proteins, gag-pol; D015683:Gene Products, gag; D011993:Recombinant Fusion Proteins; D012996:Solutions; D054301:gag Gene Products, Human Immunodeficiency Virus; C083002:p6 gag protein, Human immunodeficiency virus 1; D016333:HIV Protease