Both the cis-trans equilibrium and isomerization dynamics of a single proline amide modulate β2-microglobulin amyloid assembly.

The human protein β2-microglobulin (β2m) aggregates as amyloid fibrils in patients undergoing long-term hemodialysis. Isomerization of Pro32 from its native cis to a nonnative trans conformation is thought to trigger β2m misfolding and subsequent amyloid assembly. To examine this hypothesis, we systematically varied the free-energy profile of proline cis-trans isomerization by replacing Pro32 with a series of 4-fluoroprolines via total chemical synthesis.

Ionization state of the catalytic dyad Asp25/25' in the HIV-1 protease: NMR studies of site-specifically 13C labelled HIV-1 protease prepared by total chemical synthesis.

Total chemical synthesis was used to site-specifically (13)C-label active site Asp25 and Asp25' residues in HIV-1 protease and in several chemically synthesized analogues of the enzyme molecule. (13)C NMR measurements were consistent with a monoprotonated state for the catalytic dyad formed by the interacting Asp25, Asp25' side chain carboxyls.

Protein conformational dynamics in the mechanism of HIV-1 protease catalysis.

We have used chemical protein synthesis and advanced physical methods to probe dynamics-function correlations for the HIV-1 protease, an enzyme that has received considerable attention as a target for the treatment of AIDS. Chemical synthesis was used to prepare a series of unique analogues of the HIV-1 protease in which the flexibility of the "flap" structures (residues 37-61 in each monomer of the homodimeric protein molecule) was systematically varied. These analogue enzymes were further studied by X-ray crystallography, NMR relaxation, and pulse-EPR methods, in conjunction with molecular dynamics simulations.

Dynamics of "flap" structures in three HIV-1 protease/inhibitor complexes probed by total chemical synthesis and pulse-EPR spectroscopy.

The unliganded form of nitroxide spin-labeled HIV-1 protease and three different complexes with inhibitors were studied by pulse-EPR spectroscopy to determine "interflap" distance distributions in solution. In the unliganded enzyme, we observed a rather broad distribution with three maxima corresponding to three flap conformers; the principal form is a "semiopen/semiopen" conformer. In the complexes with inhibitors, the dominant conformer is an asymmetric "closed/semiopen" form.

Reprint of "Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site" [Bioorg. Med. Chem. Lett. 18 (2008) 4554-4557].

Here we report the X-ray structures of chemically synthesized HIV-1 protease and the inactive [D25N]HIV-1 protease complexed with the ketomethylene isostere inhibitor Ac-Thr-Ile-Nlepsi[CO-CH(2)]Nle-Gln-Arg.amide at 1.4 and 1.

Crystal structure of chemically synthesized HIV-1 protease and a ketomethylene isostere inhibitor based on the p2/NC cleavage site.

Here we report the X-ray structures of chemically synthesized HIV-1 protease and the inactive [D25N]HIV-1 protease complexed with the ketomethylene isostere inhibitor Ac-Thr-Ile-Nle psi[CO-CH(2)]Nle-Gln-Arg.amide at 1.4 and 1.

Convergent chemical synthesis and high-resolution x-ray structure of human lysozyme.

In this article, we report the total chemical synthesis of human lysozyme. Lysozyme serves as a widespread model system in various fields of biochemical research, including protein folding, enzyme catalysis, and amyloidogenesis. The 130-aa wild-type polypeptide chain of the human enzyme was assembled from four polypeptide segments by using native chemical ligation in a fully convergent fashion.