A chemometric analysis of ligand-induced changes in intrinsic fluorescence of folate binding protein indicates a link between altered conformational structure and physico-chemical characteristics.

Ligand binding alters the conformational structure and physico-chemical characteristics of bovine folate binding protein (FBP). For the purpose of achieving further information we analyzed ligand (folate and methotrexate)-induced changes in the fluorescence landscape of FBP. Fluorescence excitation and emission two-dimensional (2D) spectra were recorded over a wide range of wavelengths on a Perkin-Elmer LS 55 spectrofluorometer at varying pH in different buffers, and the resulting three-dimensional data were subjected to a chemometric analysis, parallel factor analysis (PARAFAC). The most important finding was the occurrence of two maximum intensity emission wavelengths of tryptophan, 350 nm (component one) and 330 nm (component two). In contrast to the first component, the score of the short wavelength component increased with increasing ligation of FBP. Since the emission wavelengths of indole groups in tryptophan shorten with increasing distance from the solvent surface of proteins, an increasing number of the 11 tryptophan residues seem to reorientate from the solvent surface to the interior of FBP with increasing ligation. The sharp decrease in hydrophobicity at pI=7-8 following binding of folate accords fairly well with the disappearance of strongly hydrophobic tryptophan residues from the solvent-exposed surface of FBP. The PARAFAC has thus proven useful to establish a hitherto unexplained link between parallel changes in conformational structure and physico-chemical characteristics of FBP induced by folate binding. Parameters for ligand binding derived from PARAFAC analysis of the fluorescence data were qualitatively and quantitatively similar to those obtained from binding of radiofolate to FBP. Herein, methotrexate exhibited a higher affinity for FBP than in competition with radiofolate. This could suggest a rapid and firm complexation of folate to FBP, blocking access of competing ligands.