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. Here, we devise an automated solution to the problem of multiple particles entering a region of interest, an otherwise labor-intensive and subjective process that had been performed manually in our previous work. The resolution of these two issues increases the quantity of FRET data and improves the accuracy with which FRET distributions are generated, increasing knowledge about the biological functions of the molecules under study. Our automated approach is straightforward, interpretable, and requires only localization and intensity values for donor and acceptor channel signals, which we compute through our previously published smCellFRET pipeline. The development of our automated approach is informed by the insights of expert experimentalists with extensive experience inspecting smFRET trajectories (displacement and intensity traces) from live cells. We test our automated approach against our recently published research on the metabotropic glutamate receptor 2 (mGluR2) and reveal substantial similarities, as well as potential shortcomings in the manual curation process that are addressable using the algorithms we developed here.

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. We leverage this in-cell smFRET approach to show agonist-induced structural dynamics within individual metabotropic glutamate receptor dimers. We apply these methods to representative class A, B and C receptors, finding evidence for receptor monomers, density-dependent dimers and constitutive dimers, respectively.

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. To identify key bottlenecks in this technology that can be used to guide further engineering developments, we synthesized a series of Cy5 derivatives linked to the protective agent cyclooctatetraene (COT) and examined the photophysical mechanisms curtailing their performance. The data obtained reveal that the photostability of self-healing fluorophores is limited by reactivity of the COT protective agent. The addition of electron withdrawing substituents to COT reduced its susceptibility to reactions with molecular oxygen and the fluorophore to which it is attached and increased its capacity to participate in triplet energy transfer. Exploiting these insights, we designed and synthesized a suite of modified COT-fluorophores spanning the visible spectrum that exhibited markedly increased intra-molecular photostabilization. Under ambient oxygen conditions, the photostability of Cy3 and Cy5 fluorophore derivatives increased by 3- and 9-fold in vitro and by 2- and 6-fold in living cells, respectively. We further show that this approach can improve a silicon rhodamine fluorophore. These findings offer a clear strategy for achieving the full potential of the self-healing strategy and its application to the gamut of fluorophore species commonly used for biomedical imaging.

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. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer. The time resolution of these measurements was extended to 2.5 and 10 ms, a 10- to 50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

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. Here, we address the structure-function relationship that governs accommodation using all-atom molecular simulations and single-molecule fluorescence resonance energy transfer (smFRET). Simulations that employ an all-atom, structure-based (Go-like) model illuminate the interplay between configurational entropy and effective enthalpy during the accommodation process. This delicate balance leads to spontaneous reversible accommodation attempts, which are corroborated by smFRET measurements. The dynamics about the endpoints of accommodation (the A/T and A/A conformations) obtained from structure-based simulations are validated by multiple 100-200 ns explicit-solvent simulations (3.2 million atoms for a cumulative 1.4 micros), and previous crystallographic analysis. We find that the configurational entropy of the 3'-CCA end of aminoacyl-tRNA resists accommodation, leading to a multistep accommodation process that encompasses a distribution of parallel pathways. The calculated mechanism is robust across simulation methods and protocols, suggesting that the structure of the accommodation corridor imposes stringent limitations on the accessible pathways. The identified mechanism and observed parallel pathways establish an atomistic framework for interpreting a large body of biochemical data and demonstrate that conformational changes during translation occur through a stochastic trial-and-error process, rather than in concerted lock-step motions.