Fluorescent labeling is of paramount importance to biological studies of proteins. For the development of new extrinsic small fluorophores, a series of tryptophan analogs has been designed and synthesized. Each of the analogs was used to activate a suppressor tRNA transcript and was incorporated with good efficiency into different positions of ecDHFR. The photophysical properties of the tryptophan (Trp) analogs have been examined, some of which were significantly brighter and red-shifted in fluorescence emission relative to tryptophan so that they can be selectively monitored even in the presence of multiple native tryptophan residues in the protein. Some Trp analogs form efficient FRET pairs with acceptors such as acridon-2-ylalanine (Acd) or L-(7-hydroxycoumarin-4-yl)ethylglycine (HCO) for the selective study of conformational changes in proteins. Further, 6-cyanotryptophan (6-CNTrp) was incorporated into two DNA binding proteins, including the Klenow fragment of DNA polymerase I and RNA recognition motifs (RRMs) of hnRNP LL. Using these proteins, we demonstrated the use of FRET involving 6-CNTrp as fluorescence donor and benzo[g]quinazoline-2,4-(1H, 3H)-dione 2′-deoxyriboside (Tf) or 4-aminobenzo[g]quinazoline-2-one 2′-deoxyriboside (Cf) as fluorescent acceptors to study the binding interaction of the Klenow fragment with duplex DNA oligomers (labeled with Tf), or the domain-specific association between hnRNP LL and the i-motif DNA (labeled with Cf). These findings enable a more detailed analysis in which the FRET experiments could be performed using a wide variety of experimental conditions (e.g., temperature, varying pH and salt concentration) with fluorescent donors at different locations of the Klenow fragment or RRM domains.

Tryptophan derivatives as novel fluorescent probes for studying protein conformational changes and DNA–protein interaction

253rd National Meeting (2017)

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