Monitoring of three distinct structures of restriction enzyme complexes using characteristic fluorescence from site-selectively incorporated solvatochromic probe

Abstract

The local change in the three different structures of restriction enzyme BamHI, which include DNA-free dimer and non-specific and specific complexes with DNA, were detected by the fluorescence from a site-selectively introduced solvatochromic fluorophore Nbeta-L-alanyl-5-(N,N-dimethylamino)naphthalene-1-sulfonamide (DanAla). According to the crystal structure, alpha-helices of the non-specific complex containing Ile82, Glu86 and Trp206 residues are converted into random coil by the formation of specific complex with a substrate. To understand the microenvironmental change caused by the structural transition around these positions, the DanAla probe was site-specifically introduced into the positions, and steady-state and time-resolved fluorescence was observed. The steady-state fluorescence gave us information that the rigidity of the polypeptide chains would be enhanced by the formation of the specific complex. The time-resolved fluorescence supported that the change in a water molecule-accessible space was induced by DNA-binding. We revealed that the change in rigidity and solvation around the specific positions was detected by the characteristic fluorescence using the combination of steady-state and time-resolved fluorescence techniques.