Self-association and Domains of Interactions of an Amphipathic Helix Peptide Inhibitor of HIV-1 Integrase Assessed by Analytical Ultracentrifugation and NMR Experiments in Trifluoroethanol/H2O Mixtures

Abstract

EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) is a better inhibitor of human immunodeficiency virus, type1, integrase than its parent Lys-159, reproducing the enzyme segment 147-175 with a nonpolar-polar/charged residue periodicity defined by four helical heptads (abcdefg) prone to collapse into a coiled-coil. Circular dichroism, nuclear magnetic resonance, sedimentation equilibrium, and chemical cross-linking were used to analyze EAA26 in various trifluoroethanol/H 2 O mixtures. In pure water the helix content is weak but increases regularly up to 50 -60% trifluoroethanol. In contrast the multimerization follows a bell-shaped curve with monomers in pure water, tetramers at 10% trifluoroethanol, and dimers at 40% trifluoroethanol. All suggest that interhelical interactions between apolar side chains are required for the coiled-coil formation of EAA26 and subsist at medium trifluoroethanol concentration. The N H temperature coefficients measured by nuclear magnetic resonance show that at low trifluoroethanol concentration the amide groups buried in the hydrophobic interior of four ␣-helix bundles are weakly accessible to trifluoroethanol and are only weakly subject to its hydrogen bond strengthening effect. The increased accessibility of trifluoroethanol to buried amide groups at higher trifluoroethanol concentration entails the reduction of the hydrophobic interactions and the conversion of helix tetramers into helix dimers, the latter displaying a smaller hydrophobic interface. The better inhibitory activity of EAA26 compared with Lys-159 could arise from its better propensity to form a helix bundle structure with the biologically important helical part of the 147-175 segment in integrase.The well defined conformational properties exhibited by many peptides in solution have led to their use as models for protein folding and stability but also for the design of peptide inhibitors of enzymes (1-3). The model peptides are either artificial (4 -6) or may reproduce protein segments (7-13). When they have defined conformations, the peptides can provide useful information on the relationships between local interactions, secondary structures, and tertiary structures (14 -17).We have previously tested the hypothesis that synthetic peptides reproducing amphipathic helical segments of enzymes may interact with these segments and interfere with the catalytic properties. Examples concern topoisomerase II (10, 11), an enzyme involved in the maintenance of DNA topology, and also retroviral integrase (IN) 1 (12, 13) that catalyzes the integration of viral DNA into host cellular DNA (two recent reviews, Refs. 18 and 19). IN has no cellular counterpart and can be therefore considered as a specific target for development of anti-HIV therapy (20). In an earlier study, we have reported (13) that Lys-159, a peptide corresponding to the 147-175 segment of HIV-1 IN, inhibited the integration catalyzed by IN, most likely through specific binding to its counterpart in the protein. Such a specific binding was plausible since in the crystal struct...

Probing the self‐association, intermolecular contacts, and folding propensity of amelogenin

“…1(B)]. [28][29][30] Hence, based upon the spectral difference between the AQ-and 70-rP172 samples, we conclude that TFE has a concentration-dependent effect not only on the solventrP172 NH chemical exchange rates but also the backbone conformation of amelogenin relative to the aqueous, monomeric state.…”

“…[28][29][30][31][32][33] For rP172, CD experiments performed at pH 5.8 in the presence of TFE reveal alcohol-dependent global conformational transitions [ Fig. 1(A)].…”

“…28,29 A direct overlay comparison of the 15 N-1 H HSQC AQ-and 70-rP172 spectra reveals resonant frequency mismatch arising from TFE solvent-exchange-and conformationally induced chemical shift effects. [28][29][30][31][32][33][34][35] Given that monomeric rP172 exists in a globally extended conformational state, 9 it is not surprising that this protein is susceptible to TFE -OH proton exchange [28][29][30] at NH backbone and sidechain sites. As a result, TFE and water proton chemical exchange significantly contribute to the differences in 1 H frequency dispersion [ Fig.…”

“…Over the last decade, the technique of ''solvent engineering'' 28 or the use of cosolvent mixtures of water with alcohols such as 2,2,2-trifluoroethanol (TFE) have been employed to change the environment around proteins and mimic certain conditions or to promote of protein folding and self-assembly with reasonable success. [28][29][30][31][32][33] Thus, we chose this approach to simultaneously achieve several goals that cannot be accomplished by other means with IDPs like amelogenin at the present time. These goals are: promote native-like structure within amelogenin, probe disorder-to-order transformations within the three major regions, and at TFE concentrations > 40% v/v [28][29][30][31][32] induce amelogenin oligomerization in a manner that allows identification of intermolecular contacts between amelogenin molecules.…”

“…For the first time, we report the use of TFE/ water mixtures as an extrinsic ''trigger'' 28 to promote folding within rP172 amelogenin and simultaneously promote protein self-assembly 29 under conditions that normally favor the monomeric state (i.e., low pH, low ionic strength). These experiments reveal that both the N-and C-terminal domains undergo major folding transitions and form helical structures while the central domain is largely resistant to TFE folding.…”