Ligand binding of neutral progesterone, basic propranolol, and acidic warfarin to human α1-acid glycoprotein (AGP) was investigated by Raman spectroscopy. The binding itself is characterized by a uniform conformational shift in which a tryptophan residue is involved. Slight differences corresponding to different contacts of the individual ligands inside the β-barrel are described. Results are compared with in silico ligand docking into the available crystal structure of deglycosylated AGP using quantum/molecular mechanics. Calculated binding energies are -18.2, -14.5, and -11.5 kcal/mol for warfarin, propranolol, and progesterone, respectively. These calculations are consistent with Raman difference spectroscopy; nevertheless, minor discrepancies in the precise positions of the ligands point to structural differences between deglycosylated and native AGP. Thermal dynamics of AGP with/without bounded warfarin was followed by Raman spectroscopy in a temperature range of 10-95 °C and analyzed by principal component analysis. With increasing temperature, a slight decrease of α-helical content is observed that coincides with an increase in β-sheet content. Above 45 °C, also β-strands tend to unfold, and the observed decrease in β-sheet coincides with an increase of β-turns accompanied by a conformational shift of the nearby disulfide bridge from high-energy trans-gauche-trans to more relaxed gauche-gauche-trans. This major rearrangement in the vicinity of the bridge is not only characterized by unfolding of the β-sheet but also by subsequent ligand release. Hereby, ligand binding alters the protein dynamics, and the more rigid protein-ligand complex shows an improved thermal stability, a finding that contributes to the reported chaperone-like function of AGP.

Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic Zámek 136 CZ 37333 Nové Hrady Czech Republic Faculty of Sciences University of South Bohemia Zámek 136 CZ 37333 Nové Hrady Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 5 CZ 12116 Prague 2 Czech Republic

Institute of Physics Faculty of Mathematics and Physics Charles University Prague Ke Karlovu 5 CZ 12116 Prague 2 Czech Republic Institute of Microbiology Academy of Sciences of the Czech Republic Vídeňská 1083 CZ 142 20 Prague 4 Czech Republic

Citace poskytuje Crossref.org