title: Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase. Asymmetry in p66 subunits of the p66/p66 homodimer.
abstract: A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can be obtained [(1991) Biotechnol. Appl. Biochem. 14, 69-81] from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC). We have analyzed the p66 HIV-1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12. The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak). Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4 degrees C. Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min. The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease. When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC. While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A. These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.
authors.affiliation: Upjohn Laboratories, Kalamazoo, MI 49002.
authors.forename: S K
authors.initials: SK
authors.lastname: Sharma
authors.forename: N
authors.initials: N
authors.lastname: Fan
authors.forename: D B
authors.initials: DB
authors.lastname: Evans
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D011487:Protein Conformation; D017510:Protein Folding; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H