Single-Molecule Fluorescence and FRET Measurements on Internalized Proteins in Living Bacteria

Despite advances in structural biology techniques (such as NMR and X-ray crystallography), it is still extremely difficult to recover information on protein structure, conformations and dynamics inside living cells. Fluorescent protein fusions provide an avenue to study proteins in vivo but cannot directly report on protein structure due to limitations in size, labeling position, and photophysical properties; as a result, there is a pressing need for new methods that can study protein structure in vivo.Towards this goal, we developed electroporation methods to internalize proteins labeled with organic fluorophores in living E. coli. Electroporated cells retained viability while being loaded with different singly or doubly labeled proteins of various sizes (15-100 kDa) and observed at the single-cell and single-molecule levels; single-color fluorescence or FRET could be observed for several seconds. The number of internalized proteins could be tuned from a few to several hundred copies per cell.We used the diffusion behavior of internalized DNA-binding proteins to study protein activity and cellular localization. Internalized catabolite activator protein (a bacterial activator) remains immobile in cells, likely due to binding to chromosomal sites. In contrast, internalized DNA polymerase I Klenow fragment (KF), a component of DNA repair pathways, diffuses through the cytoplasm and localizes when DNA damaging reagents are added. Measurements on doubly-labeled KF molecules reveal FRET efficiencies centered ∼50% (corresponding to an interprobe distance of ∼5 nm), matching published in vitro results, and enabling the study of KF conformations and dynamics in vivo. Ongoing work focuses on internalization of partners of specific complexes to examine their structure and interactions in the bacterial cytoplasm, as well as internalization in other organisms.