Raman spectroscopic studies of ligand-protein interactions: the binding of biotin analogues by avidin

Abstract

The binding of avidin (Avi) with biotin (Bio) and some Bio analogues (biotin methyl ester, desthiobiotin, 2′-iminobiotin and diaminobiotin) was studied by means of Fourier transform Raman spectroscopy. The difference in affinity between the protein and these ligands provides a detailed picture of the effects of modification of the Bio structure on the binding strength in the complex. The vibrational results from both qualitative and quantitative analysis indicate that as a consequence of the interaction with the ligands, the content of the β-sheet conformation decreases whereas that of the α-helix conformation increases. To confirm further the conformational changes of the protein structure due to the Bio analogues’ binding, curve-fitting analysis of the amide I Raman band of Avi alone and of the complexes was performed. The fourth derivatives of the amide I band of the spectra examined (1630–1700 cm-1 region) were analyzed in order to determine the starting data required for the curve-fitting procedure. The ratios between the integrated intensities of the β-sheet and α-helix components, calculated for each system, confirm the results obtained by means of the quantitative analysis of the amide I Raman band. A good correlation was found between the spectroscopic results and the affinity of each ligand toward Avi (∣r∣⩾0.91). This linear relationship between the secondary structure percentages and pKD of the complexes allows for the evaluation of the dissociation constants of the Avi complexes. The present study confirms the participation of many factors in the stability of the protein complexes. The Bio molecule is arranged in the active site in such a way that an extended hydrogen bond network, able to stabilize the complex, can be established. Trp residues do not directly bind the ligand and contribute to the formation of the complex by means of their hydrophobic interactions. © 1998 John Wiley & Sons, Ltd.