Monitoring of three distinct structures of restriction enzyme complexes using characteristic fluorescence from site-selectively incorporated solvatochromic probe.
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.
The Institute of Scientific and Industrial Research, Osaka University Mihogaoka 8-1, Ibaraki, Osaka, 567-0047, Japan.
Koji
K
Nakayama
Masayuki
M
Endo
Mamoru
M
Fujitsuka
Tetsuro
T
Majima
D004262:DNA Restriction Enzymes / Q000737:chemistry*; D005453:Fluorescence; D008958:Models, Molecular; D015335:Molecular Probes; D011487:Protein Conformation
D015335:Molecular Probes; D004262:DNA Restriction Enzymes