Conformational stability of dimeric HIV-1 and HIV-2 reverse transcriptases.
The dissociation of dimeric reverse transcriptase (RT) of the human immunodeficiency virus (HIV) types 1 and 2 has been investigated using acetonitrile as a dissociating agent. The equilibrium transitions were monitored by combining different approaches (fluorescence spectroscopy, polymerase activity assay, and size-exclusion HPLC). The dissociation of RT induced a complete loss of polymerase activity and a 25% increase of the intrinsic fluorescence. It is fully reversible, and the midpoints of the equilibrium transition curves are dependent on the concentration of the enzyme used, suggesting a two-state transition model for the dissociation of RT in which dimers are in equilibrium with folded monomers. For both RTs, the heterodimeric form is more stable against dissociating agents and different pH than the corresponding homodimeric form. Moreover, heterodimeric HIV-2 RT exhibits a higher stability than HIV-1 RT, with a free energy of dissociation of 12.1 kcal/mol at pH 6.5 and 25 degrees C, instead of 10 kcal/mol for HIV-1 RT. The binding of a primer/template induces a marked conformational change in both RTs, shown by the lower accessibility of the tryptophans to quenchers and the increase in tryptophan heterogeneity, and stabilized the dimeric form of both RTs (10-100-fold). The central role of hydrophobic interactions in dimer formation has been revealed by the 30% increase of exposure of the tryptophan cluster to quenchers upon dissociation of RT and the binding of 4 equiv of 1-anilino-8-naphthalenesulfonate to the dissociated enzymes.
Divita
G
G
Max-Planck-Institut für Medizinische Forschung, Abteilung Biophysik, Heidelberg, Germany.
Rittinger
K
K
Restle
T
T
Immendörfer
U
U
Goody
R S
RS
D000097:Acetonitriles / Q000494:pharmacology; D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D017931:DNA Primers; D004795:Enzyme Stability; D005456:Fluorescent Dyes / Q000378:metabolism; D054303:HIV Reverse Transcriptase; D015497:HIV-1 / Q000201:enzymology*; D015498:HIV-2 / Q000201:enzymology*; D007454:Iodides / Q000494:pharmacology; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics
D000097:Acetonitriles; D000178:Acrylamides; D000817:Anilino Naphthalenesulfonates; D017931:DNA Primers; D005456:Fluorescent Dyes; D007454:Iodides; D020106:Acrylamide; C027132:1-anilino-8-naphthalenesulfonate; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); C072416:reverse transcriptase, Human immunodeficiency virus 2; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; C032159:acetonitrile