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 fluorescence polarization of both ANS and bis-ANS is excitation wavelength dependent and this is a property of all protein-ANS/bis-ANS complexes studied. Bis-ANS excitation maximum is always more red shifted than the corresponding ANS complex. Even when corrected for the red shift, the bis-ANS complexes in some, but not all, cases show only a little lowering 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 values of the naphthyl-naphthyl dihedral angle. Although, Molecular mechanics and dynamics calculations show that the lowest energy conformation of bis-ANS is when the two naphthalene rings are roughly perpendicular to each other, due to rapid energy migration this conformation should lead to dramatic lowering of emission anisotropy, unlike what is observed. Salt and temperature dependence of bis-ANS/protein interaction suggests little ionic interaction and pre-dominant interaction through hydrophobic aromatic rings. We conclude that bis-ANS binds to proteins through interaction with the aromatic rings and with two rings nearly parallel to each other.
Bothra
A
A
Department of Biophysics, Bose Institute, Calcutta, India.
Bhattacharyya
A
A
Mukhopadhyay
C
C
Bhattacharyya
K
K
Roy
S
S
D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates / Q000737:chemistry* / Q000378:metabolism*; D016880:Anisotropy; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism*; D015394:Molecular Structure; D011506:Proteins / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013816:Thermodynamics
D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; D011506:Proteins; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D000604:Amino Acyl-tRNA Synthetases; C020659:glutaminyl-tRNA synthetase