Use of protein unfolding studies to determine the conformational and dimeric stabilities of HIV-1 and SIV proteases.
The free energies of dimer dissociation of the retroviral proteases (PRs) of human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus (SIV) were determined by measuring the effects of denaturants on the protein fluorescence upon the unfolding of the enzymes. HIV-1 PR was more stable to denaturation by chaotropes and extremes of pH and temperature than SIV PR, indicating that the former enzyme has greater conformational stability. The urea unfolding curves of both proteases were sigmoidal and single phase. The midpoints of the transition curves increased with increasing protein concentrations. These data were best described by and fitted to a two-state model in which folded dimers were in equilibrium with unfolded monomers. This denaturation model conforms to cases in which protein unfolding and dimer dissociation are concomitant processes in which folded monomers do not exist [Bowie, J. U., & Sauer, R. T. (1989) Biochemistry 28, 7140-7143]. Accordingly, the free energies of unfolding reflect the stabilities of the protease dimers, which for HIV-1 PR and SIV PR were, respectively, delta GuH2O = 14 +/- 1 kcal/mol (Ku = 39 pM) and 13 +/- 1 kcal/mol (Ku = 180 pM). The binding of a tight-binding, competitive inhibitor greatly stabilized HIV-1 PR toward urea-induced unfolding (delta GuH2O = 19.3 +/- 0.7 kcal/mol, Ku = 7.0 fM). There were also profound effects caused by adverse pH on the protein conformation for both HIV-1 PR and SIV PR, resulting in unfolding at pH values above and below the respective optimal ranges of 4.0-8.0 and 4.0-7.0
Grant
S K
SK
Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939.
Deckman
I C
IC
Culp
J S
JS
Minnich
M D
MD
Brooks
I S
IS
Hensley
P
P
Debouck
C
C
Meek
T D
TD
D016282:Aspartic Acid Endopeptidases / Q000737:chemistry* / Q000187:drug effects; D004795:Enzyme Stability / Q000187:drug effects; D016333:HIV Protease / Q000737:chemistry* / Q000187:drug effects; D015497:HIV-1 / Q000201:enzymology*; D006358:Hot Temperature; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary / Q000187:drug effects; D011994:Recombinant Proteins / Q000737:chemistry / Q000187:drug effects; D015302:Simian Immunodeficiency Virus / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014461:Ultracentrifugation; D014508:Urea / Q000494:pharmacology
D011994:Recombinant Proteins; D014508:Urea; D016282:Aspartic Acid Endopeptidases; D016333:HIV Protease