Human a₁-acid glycoprotein investigated by Raman spectroscopy and Raman optical activity

V. Kopecký Jr.¹, K. Hofbauerová^{1, 2}, R. Ettrich³, J. Kapitán¹, V. Baumruk¹

¹Institute of Physics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 5, Prague 2, CZ-12116, Czech Republic

²Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4, CZ-14220, Czech Republic

³Laboratory of High Performance Computing, Institute of Physical Biology of USB and Institute of Systems Biology and Ecology of AS CR, Zámek 136, Nové Hrady, CZ-37333, Czech Republic

kopecky@karlov.mff.cuni.cz

a₁-Acid glycoprotein (AGP), also known as orosomucoid, contains 183 amino acids with 21 possible substitutions in one polypeptide chain linked by two disulfide bonds. The content of carbohydrate moiety is slightly above 40% of the total molecular weight of 41 kDa of the protein. Five branched carbohydrate units terminated by sialic acid residues are linked to Asn residues of the peptide chain. It is known that AGP plays a role under inflammatory or other pathological conditions and is able to bind basic drugs and certain steroid hormones [1]. However, the exact role of AGP as well as its 3D structure is not entirely clear.

A model of 3D structure of AGP has been proposed by an approach that combines molecular modeling and vibrational spectroscopy [2]. It shows that AGP folds as a symmetrical all-b protein dominated by an eight-stranded antiparallel b-sheet. Binding of progesterone is described with a coincident transformation of the a-helix above the binding pocket into antiparallel b-sheet [2]. Here we investigated ligand binding of AGP followed by three ligands (natural and artificial) with different physico-chemical properties, i.e. progesterone, propranolol and warfarin. Raman difference spectroscopy shows the same pattern upon binding of all ligands which suggest presence of only one high-affinity binding site in AGP. The spectral changes reflect a presence of Trp¹²² in the binding site as was proposed by molecular modeling and dynamics simulations. The ligand release must be connected with a reverse structural behavior where the carbohydrate moiety in interaction with membranes could play a role. Therefore, we investigated structure of AGP with respect to carbohydrate moiety and its potential role in ligand binding/release by Raman optical activity (ROA). It was demonstrated, on the case of N-acetyl neuraminic acid, that ROA spectra of carbohydrates are very useful in interpretations of ROA bands of glycoproteins.

References

1. T. Fournier, N. Medjoubi-N, D. Porquet, Biochim. Biophys. Acta,1482, (2000), 157.

2. V. Kopecký Jr., R. Ettrich, K. Hofbauerová, V. Baumruk, Biochem. Biophys. Res. Commun., 300, (2003), 41.

Acknowledgements

The Grant Agency of the Czech Republic and the Ministry of Education of the Czech Republic are gratefully acknowledged for support (No. 202/06/P208 and No. MSM 0021620835, respectively).