Fluorophore-quencher pair for monitoring protein motion.

ABSTRACT: A fluorophore/quencher pair capable of detecting conformational changes of DNA-protein complexes is described. The system employs a fluorescent nucleoside analog 1,3-diaza-2-oxophenothiazine (tC) within duplex DNA and a non-fluorescent quencher (TEMPO) attached to an engineered cysteine residue of the protein. The straightforward labeling methodology allows for the placement of the fluorophore and quencher moieties at specific positions suited to studying the conformational change of interest. To illustrate the utility of the tC-TEMPO pair, we have monitored nucleotide-induced conformational changes of the Klenow fragment (KF) polymerase bound to duplex DNA. In this system, tC was incorporated in the primer strand of the duplex, adjacent to the 3' end, while TEMPO was positioned at the end

SUBMITTER: Tahmassebi DC 

PROVIDER: S-EPMC2693715 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature