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 enzymeBamHI, 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 N^β-L-alanyl-5-(N,N-dimethylamino)naphthalene-1-sulfonamide (DanAla). According to the crystal structure, α-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.