title: Mutational and structural studies aimed at characterizing the monomer of HIV-1 protease and its precursor.
abstract: An experimental protocol for folding the mature human immunodeficiency virus-1 (HIV-1) protease is presented that facilitates NMR studies at a low protein concentration of approximately 20 micoM. Under these conditions, NMR spectra show that the mature protease lacking its terminal beta-sheet residues 1-4 and 96-99 (PR(5-95)) exhibits a stable monomer fold spanning the region 10-90 that is similar to that of the single subunit of the wild-type dimer and the dimer bearing a D25N mutation (PR(D25N)). Urea-induced unfolding monitored both by changes in (1)H-(15)N heteronuclear single quantum correlation spectra and by protein fluorescence indicates that although PR(5-95) monomer displays a transition profile similar to that of the PR(D25N) dimer (50% unfolded (U(50)) = approximately 1.9 M), extending the protease with 4 residues (SFNF) of its N-terminally flanking sequence in the Gag-Pol precursor ((SFNF)PR(D25N)) decreases the stability of the fold (U(50) = approximately 1.5 M). Assigned backbone chemical shifts were used to elucidate differences in the stability of the PR(T26A) (U(50) = 2.5 M) and (SFNF)PR(D25N) monomers and compared with PR(D25N/T26A) monomer. Discernible differences in the backbone chemical shifts were observed for N-terminal protease residues 3-6 of (SFNF)PR(D25N) that may relate to the increase in the equilibrium dissociation constant (K(d)) and the very low catalytic activity of the protease prior to its autoprocessing at its N terminus from the Gag-Pol precursor.
authors.affiliation: Department of Structural Biology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
authors.forename: Rieko
authors.initials: R
authors.lastname: Ishima
authors.forename: Dennis A
authors.initials: DA
authors.lastname: Torchia
authors.forename: John M
authors.initials: JM
authors.lastname: Louis
mesh_terms: D019281:Dimerization; D005453:Fluorescence; D016333:HIV Protease / Q000737:chemistry* / Q000235:genetics*; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D014508:Urea / Q000737:chemistry
chemical_list: D014508:Urea; D016333:HIV Protease