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Large DNA molecules were visualized with novel fluorescent protein–DNA binding peptides (FP–DBPs). We constructed FP–DBPs by linking fluorescent protein to the N- or C-terminal of one or two peptides. We designed these DNA binding peptides from various DNA binding motifs such as oligo-lysine (K) and Lys-Trp (KW) repeats, TPKRPRGRPKK from high mobility group (HMG) chromosomal protein, and Ser-Pro-Arg-Lys (SPRK) from histone protein. We demonstrated the use of FP–DBP to stain large DNA molecules, and then analysed the fluorescence brightness and their binding affinity to double stranded DNA. This investigation provided HMG-tagged FP–DBP as the best DNA staining reagent in terms of fluorescence intensity, signal-to-noise ratio, and DNA binding affinity (Kd = 586 nM). Furthermore, we measured elongation of FP–DBP-stained DNA molecules tethered on the surface in order to evaluate FP–DBP-induced structural deformation.

FRET sensors based on fluorescent proteins have been powerful tools for probing protein–protein interactions and structural changes within proteins. However, they are intrinsically limited by their large size and the requirement for N- or C-terminal fusions. In this report, a FRET-based sensor was developed by incorporating a fluorescent unnatural amino acid into glutamine-binding protein (GlnBP), which formed a FRET pair with green fluorescent protein (GFP). GFP was fused to the N-terminus of GlnBP, and the fluorescent unnatural amino acid was incorporated into the site for N138 in GlnBP. FRET sensors were tested that contained linkers of different length between GFP and GlnBP, and assay conditions were optimized by changing the pH and salt concentration of the assay buffer. Under optimal conditions, the best sensor protein produced a 1.9-fold increase in the FRET ratio upon L-Gln binding, whereas either no change or minimal change was seen using other amino acids, including the other 19 natural amino acids and D-Gln. This novel design strategy for FRET sensors overcomes the limitations of current FRET sensors, which require the use of two fluorescent proteins. Consequently, our strategy may prove useful for investigating protein–protein interactions and for probing changes in protein conformation.

Four unnatural amino acids (UAAs) were synthesized from their corresponding α-keto acids by using glutamine:phenylpyruvate aminotransferase (GlnAT) from Thermus thermophilus HB8. The enzyme efficiently catalyzed the conversion of the unnatural substrates, with little decrease in its activity compared to that shown toward its natural substrates. The α-keto acids were converted into the corresponding amino acids in cells and then directly incorporated into a protein with high efficiency and fidelity. Because α-keto acids have no chiral center and are synthetically readily accessible, this method will further enhance the genetic incorporation technology and may present a new approach to the preparation of optically pure UAAs. Considering the substrate promiscuity of the GlnAT observed in the study, this method can be applied to the genetic incorporation of other UAAs derived from Tyr or Phe, which constitute approximately 50% of the successfully incorporated UAAs. In addition, this study provides an impetus for the development of an autonomous unnatural organism that can synthesize and incorporate UAAs.