A FLUORESCENCE SPECTROSCOPIC AND MOLECULAR-DYNAMICS STUDY OF BIS-ANS PROTEIN INTERACTION/

Despite emergence of bis-ANS as a major fluorescence probe of proteins structure, conformational and spectroscopic properties of protein/bis -ANS complexes remains largely unexplored. We have shown that fluoresc ence polarization of both ANS and bis-ANS is excitation wavelength dep endent and this is a property of all protein-ANS/bis-ANS complexes stu died. Bis-ANS excitation maximum is always more red shifted than the c orresponding ANS complex. Even when corrected for the red shift, the b is-ANS complexes in some, but not all, cases show only a little loweri ng of polarization, suggesting modest additional depolarization in bis -ANS compared to ANS. Calculation of energy migration rate between the two rings suggests that energy migration rate should be high at all v alues of the naphthyl-naphthyl dihedral angle. Although, Molecular mec hanics and dynamics calculations show that the lowest energy conformat ion of bis-ANS is when the two naphthalene rings are roughly perpendic ular to each other, due to rapid energy migration this conformation sh ould lead to dramatic lowering of emission anisotropy, unlike what is observed. Salt and temperature dependence of bis-ANS/protein interacti on suggests little ionic interaction and pre-dominant interaction thro ugh hydrophobic aromatic rings. We conclude that bis-ANS binds to prot eins through interaction with the aromatic rings and with two rings ne arly parallel to each other.