Circular dichroism studies of the HIV-1 Rev protein and its specific RNA binding site.
The circular dichroism (CD) spectrum of the Rev protein from HIV-1 indicates that Rev contains about 50% alpha helix and 25% beta sheet at 5 degrees C in potassium phosphate buffer, pH 3, and 300 mM KF. The spectrum is independent of protein concentration over a 20-fold range. At neutral pH, Rev is relatively insoluble but can be brought into solution by binding to its specific RNA binding site, the Rev-responsive element (RRE), at a Rev:RNA ratio of about 3:1. Nonspecific binding to tRNA does not solubilize Rev. As judged by difference CD spectra, the conformation of Rev when bound to the RRE at neutral pH is similar to the conformation of unbound Rev at pH 3, although changes in the RNA may also contribute to the difference spectrum. Indeed, some difference is observed near 260 nm, consistent with a conformational change of the RRE upon Rev binding. Rev alone at pH 3 shows irreversible aggregation as the temperature is raised, while Rev bound to the RRE at neutral pH shows a reversible transition with a Tm of 68 degrees C.
Daly
T J
TJ
Repligen Corporation, Cambridge, Massachusetts 02139.
Rusche
J R
JR
Maione
T E
TE
Frankel
A D
AD
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D015694:Gene Products, rev / Q000378:metabolism*; D015752:Genes, env; D015497:HIV-1 / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D012323:RNA Processing, Post-Transcriptional; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus
D002352:Carrier Proteins; D015694:Gene Products, rev; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D054321:rev Gene Products, Human Immunodeficiency Virus