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作者 Torbeev, Vladimir Y
書名 Protein dynamics and function are correlated in the chemical mechanism of HIV-1 protease catalysis
國際標準書號 9781109212419
book jacket
說明 238 p
附註 Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3497
Adviser: Stephen B. H. Kent
Thesis (Ph.D.)--The University of Chicago, 2009
Total chemical synthesis was used to prepare a series of unique chemical analogues of HIV-1 protease, where we systematically substituted the residues Gly51/51' at the tips of the mobile 'flaps' (residues 37-61 in each domain of the protein homodimer) with L-Ala, D-Ala in both symmetric and asymmetric fashion. Such substitutions, although in regions distant from the catalytic aspartates, led in most cases to a reduction of catalytic activity. In contrast to this, a 'covalent dimer' with L-Ala51 in one flap and D-A1a51' in another flap has shown native-like enzyme activity
Chemical protein synthesis was also used to prepare site-specifically 15N-isotope labeled enzymes for NMR dynamics experiments, and nitroxide spin-labelled analogues for pulse-EPR studies of protein dynamics. These spectroscopic studies provided strong evidence for critical differences in dynamic properties of the L-Ala51-containing flap versus the D-A1a51'-containing flap, with the latter being more rigid both on the sub-ns and mus-ms time scales. Using NMR relaxation-dispersion measurements we also detected coupling between the dynamics of the flaps and the catalytic aspartate residues of the HIV-1 protease. Molecular dynamics simulations suggest that the asymmetric enzyme analogue with L-Ala/D-Ala-containing flaps, but not the symmetric L-Ala/L-Ala or D-Ala/D-Ala enzymes, uniquely stabilizes the productive conformational state of catalytic residues Asp25 and Asp25', with the nucleophilic water molecule being preorganized for catalysis. Our results suggest that the homodimeric viral HIV-1 protease and the single polypeptide, inherently asymmetric, cell-encoded aspartic proteases share the same asymmetric catalytic mechanism
A low-barrier hydrogen bond (LBHB) between two catalytic residues Asp25 and Asp25' was postulated to mediate proton transfer. Experimental evidence for the existence of LBHB comes from very short O...O (Asp25...Asp25') distances (2.3-2.5 A) detected by X-ray crystallography, although electron density for the putative proton was not observed directly. We have, for the first time, detected low-barrier hydrogen bonds in HIV-1 protease/inhibitor complexes by NMR method and further provided a model of how protein dynamics is correlated to proton transfer
School code: 0330
Host Item Dissertation Abstracts International 70-06B
主題 Chemistry, Biochemistry
0487
Alt Author The University of Chicago
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