Combining Accurate FRET and Tracking of Single Protein and DNA Molecules in Live Bacteria

Abstract

In vivo single-molecule FRET (smFRET) is an excellent tool for studying nanoscale structure and conformational changes in living cells. We have recently introduced an electroporation-based method to internalize DNA and proteins labeled with organic fluorophores into living bacteria and established the ability for long-lived single-molecule fluorescence and FRET measurements. However, further developments, such as optimization of electroporation conditions as well as quantitative smFRET analysis, are needed to make the method more robust and general.Using singly labeled DNA fragments, we optimized internalization efficiency and cell viability for six electroporation voltages, achieving >70% loading and viability similar to non-treated cells. To characterize and optimize in vivo smFRET measurements, we used DNAs and proteins site-specifically labeled with organic fluorophores. Specifically, we used doubly-labeled DNA FRET standards (45-bp long and having in vitro FRET efficiencies of ∼20%, ∼50%, and ∼85%) and doubly labeled derivatives of DNA Polymerase I. Using an alternating laser excitation scheme, we measured apparent FRET efficiencies (E∗) at the single-cell and single-molecule levels for DNAs labeled with more than six FRET-dye-pairs; we also established in vivo FRET corrections for immobilized molecules accounting for cellular autofluorescence, spectral cross-talk and differences in detector efficiencies and quantum yields. We measured smFRET efficiencies within diffusing DNAs and proteins tracked for ∼10s. We have also studied the diffusion profile and FRET status of DNA Polymerase I during DNA replication and repair; the diffusion profile reports on polymerase binding to chromosomal DNA, whereas FRET efficiencies report on protein conformational states (E∗∼50%: binary polymerase-DNA complex; E∗∼70%: ternary polymerase-DNA-nucleotide complex). These novel and general smFRET tools should allow visualization of protein structure in vivo and report on how conformational changes affect cellular mechanisms.