Fluorescent dyes, attached to proteins are widely used to study different biological processes. However, physical mechanisms of fluorescence change in response to particular stimuli are poorly understood. Here we use two-photon polarization ratio Omega and cross section spectra to study the nature of local interactions between tetramethyrhodamine-6-maleimide and voltage sensing phosphatase (VSP). We show that when rhodamine binds to VSP, it undergoes a conformational change corresponding to a twist of a dihedral angle between the phenyl and xanthene planes from 90 to 70–80 degree. This change is probably due to steric clashes between the rhodamine and neighbor amino acid residues.
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