Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1.
Proteolytic experiments in conjunction with 1H-NMR spectroscopy show that the Nef (negative factor) protein from human immunodeficiency virus type 1 probably consists of two main domains, the N-terminal anchor domain at amino acid positions 2-65 and the C-terminal core domain at positions 66-206. The N-terminal domain is likely to be located at the surface of the protein, while the C-terminal domain has a compactly folded core and is stable in the absence of the anchor domain. It is conceivable that the core domain represents a functional domain of the Nef protein, activated after the removal of the membrane anchor by the human-immunodeficiency-virus protease or cellular proteases. Nef is stable at pH 5-12 and denatures at 317-322 K. The Nef protein remains in its native conformation in dimethyl-sulfoxide/water mixtures up to 35% (by vol.), and in acetonitrile/water up to 14% (by vol.). Nef refolds spontaneously after denaturation with urea or guanidinium hydrochloride. The 1H-NMR parameters and pKa values of five of the nine histidine residues and one of the seven tyrosine residues were determined and were found in four cases to be typical for residues which are not located in the interior of the protein.
Freund
J
J
Max-Planck-Institute for Medical Research, Department of Biophysics, Heidelberg, Germany.
Kellner
R
R
Houthaeve
T
T
Kalbitzer
H R
HR
D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015693:Gene Products, nef / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016333:HIV Protease / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010196:Pancreatic Elastase / Q000378:metabolism; D011489:Protein Denaturation; D012995:Solubility; D013696:Temperature; D014357:Trypsin / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D015693:Gene Products, nef; D054311:nef Gene Products, Human Immunodeficiency Virus; D014443:Tyrosine; D006639:Histidine; D010196:Pancreatic Elastase; D014357:Trypsin; D016333:HIV Protease