STEADY-STATE AND TIME-RESOLVED FLUORESCENCE QUENCHING WITH TRANSITION METAL IONS AS SHORT-DISTANCE PROBES FOR PROTEIN CONFORMATION

摘要

A series of model dye-labeled histidine-containing peptides was used to investigate the nature of the quenching mechanism with Cu²⁺ and Ni²⁺. The strong reduction in steady-state fluorescence was found to be unaccompanied by any noticeable changes in lifetime kinetics. This static nature of quenching is not consistent with the dynamic FRET phenomenon, which was assumed to dominate the quenching mechanism, and is likely caused by shorter range orbital coupling. Our results indicate that the FRET-like 6th power of distance dependence of quenching cannot be automatically assumed for transition metal ions, and time-resolved measurements should be used to distinguish various quenching mechanisms.