Methods for automating the analysis of live-cell single-molecule FRET data.

Single-molecule FRET (smFRET) is a powerful imaging platform capable of revealing dynamic changes in the conformation and proximity of biological molecules. The expansion of smFRET imaging into living cells creates both numerous new research opportunities and new challenges. Automating dataset curation processes is critical to providing consistent, repeatable analysis in an efficient manner, freeing experimentalists to advance the technical boundaries and throughput of what is possible in imaging living cells.

Single-molecule FRET imaging of GPCR dimers in living cells.

Class C G protein-coupled receptors (GPCRs) are known to form stable homodimers or heterodimers critical for function, but the oligomeric status of class A and B receptors, which constitute >90% of all GPCRs, remains hotly debated. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful approach with the potential to reveal valuable insights into GPCR organization but has rarely been used in living cells to study protein systems. Here, we report generally applicable methods for using smFRET to detect and track transmembrane proteins diffusing within the plasma membrane of mammalian cells.

Electronic tuning of self-healing fluorophores for live-cell and single-molecule imaging.

Bright, long-lasting organic fluorophores enable a broad range of imaging applications. "Self-healing" fluorophores, in which intra-molecularly linked protective agents quench photo-induced reactive species, exhibit both enhanced photostability and biological compatibility. However, the self-healing strategy has yet to achieve its predicted potential, particularly in the presence of ambient oxygen where live-cell imaging studies must often be performed.

Cyanine fluorophore derivatives with enhanced photostability.

Fluorescence applications requiring high photostability often depend on the use of solution additives to enhance fluorophore performance. Here we demonstrate that the direct or proximal conjugation of cyclooctatetraene (COT), 4-nitrobenzyl alcohol (NBA) or Trolox to the cyanine fluorophore Cy5 dramatically enhanced fluorophore photostability without otherwise affecting its native spectral characteristics. Such conjugation is a powerful means of improving the robustness of fluorescence-based applications demanding long-lived, nonblinking fluorescence emission.

Conformational sampling of aminoacyl-tRNA during selection on the bacterial ribosome.

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with elongation factor-Tu and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and, consequently, the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of elongation factor-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation.

Accommodation of aminoacyl-tRNA into the ribosome involves reversible excursions along multiple pathways.

The ribosome is a massive ribonucleoprotein complex ( approximately 2.4 MDa) that utilizes large-scale structural fluctuations to produce unidirectional protein synthesis. Accommodation is a key conformational change during transfer RNA (tRNA) selection that allows movement of tRNA into the ribosome.