RESUMEN
Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to expand the chemical space available to biological therapeutics and materials, but existing technologies are still limiting. Addressing these limitations requires a deeper understanding of the mechanism of protein synthesis and how it is perturbed by nnAAs. Here we examine the impact of nnAAs on the formation and ribosome utilization of the central elongation substrate: the ternary complex of native, aminoacylated tRNA, thermally unstable elongation factor, and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer measurements, we reveal that both the (R)- and (S)-ß2 isomers of phenylalanine (Phe) disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by 1 order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of translocation after mRNA decoding. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include the consideration of the efficiency and stability of ternary complex formation.
RESUMEN
Single-molecule fluorescence resonance energy transfer (smFRET) methods employed to quantify time-dependent compositional and conformational changes within biomolecules require elevated illumination intensities to recover robust photon emission streams from individual fluorophores. Here we show that outside the weak-excitation limit, and in regimes where fluorophores must undergo many rapid cycles of excitation and relaxation, non-fluorescing, excitation-induced triplet states with lifetimes orders of magnitude longer lived than photon-emitting singlet states degrade photon emission streams from both donor and acceptor fluorophores resulting in illumination-intensity-dependent changes in FRET efficiency. These changes are not commonly taken into consideration; therefore, robust strategies to suppress excited state accumulations are required to recover accurate and precise FRET efficiency, and thus distance, estimates. We propose both robust triplet state suppression and data correction strategies that enable the recovery of FRET efficiencies more closely approximating true values, thereby extending the spatial and temporal resolution of smFRET.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , Transferencia Resonante de Energía de Fluorescencia/métodos , Fotones , Colorantes Fluorescentes/química , Imagen Individual de Molécula/métodosRESUMEN
Templated synthesis of proteins containing non-natural amino acids (nnAAs) promises to vastly expand the chemical space available to biological therapeutics and materials. Existing technologies limit the identity and number of nnAAs than can be incorporated into a given protein. Addressing these bottlenecks requires deeper understanding of the mechanism of messenger RNA (mRNA) templated protein synthesis and how this mechanism is perturbed by nnAAs. Here we examine the impact of both monomer backbone and side chain on formation and ribosome-utilization of the central protein synthesis substate: the ternary complex of native, aminoacylated transfer RNA (aa-tRNA), thermally unstable elongation factor (EF-Tu), and GTP. By performing ensemble and single-molecule fluorescence resonance energy transfer (FRET) measurements, we reveal the dramatic effect of monomer backbone on ternary complex formation and protein synthesis. Both the (R) and (S)-ß2 isomers of Phe disrupt ternary complex formation to levels below in vitro detection limits, while (R)- and (S)-ß3-Phe reduce ternary complex stability by approximately one order of magnitude. Consistent with these findings, (R)- and (S)-ß2-Phe-charged tRNAs were not utilized by the ribosome, while (R)- and (S)-ß3-Phe stereoisomers were utilized inefficiently. The reduced affinities of both species for EF-Tu ostensibly bypassed the proofreading stage of mRNA decoding. (R)-ß3-Phe but not (S)-ß3-Phe also exhibited order of magnitude defects in the rate of substrate translocation after mRNA decoding, in line with defects in peptide bond formation that have been observed for D-α-Phe. We conclude from these findings that non-natural amino acids can negatively impact the translation mechanism on multiple fronts and that the bottlenecks for improvement must include consideration of the efficiency and stability of ternary complex formation.
RESUMEN
In all species, ribosomes synthesize proteins by faithfully decoding messenger RNA (mRNA) nucleotide sequences using aminoacyl-tRNA substrates. Current knowledge of the decoding mechanism derives principally from studies on bacterial systems1. Although key features are conserved across evolution2, eukaryotes achieve higher-fidelity mRNA decoding than bacteria3. In human, changes in decoding fidelity are linked to ageing and disease and represent a potential point of therapeutic intervention in both viral and cancer treatment4-6. Here we combine single-molecule imaging and cryogenic electron microscopy methods to examine the molecular basis of human ribosome fidelity to reveal that the decoding mechanism is both kinetically and structurally distinct from that of bacteria. Although decoding is globally analogous in both species, the reaction coordinate of aminoacyl-tRNA movement is altered on the human ribosome and the process is an order of magnitude slower. These distinctions arise from eukaryote-specific structural elements in the human ribosome and in the elongation factor eukaryotic elongation factor 1A (eEF1A) that together coordinate faithful tRNA incorporation at each mRNA codon. The distinct nature and timing of conformational changes within the ribosome and eEF1A rationalize how increased decoding fidelity is achieved and potentially regulated in eukaryotic species.
Asunto(s)
Bacterias , Biosíntesis de Proteínas , Humanos , Bacterias/genética , Bacterias/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Aminoacil-ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/metabolismo , Imagen Individual de Molécula , Microscopía por Crioelectrón , Ribosomas/genética , Ribosomas/metabolismoRESUMEN
Peptide-chain elongation during protein synthesis entails sequential aminoacyl-tRNA selection and translocation reactions that proceed rapidly (2-20 per second) and with a low error rate (around 10-3 to 10-5 at each step) over thousands of cycles1. The cadence and fidelity of ribosome transit through mRNA templates in discrete codon increments is a paradigm for movement in biological systems that must hold for diverse mRNA and tRNA substrates across domains of life. Here we use single-molecule fluorescence methods to guide the capture of structures of early translocation events on the bacterial ribosome. Our findings reveal that the bacterial GTPase elongation factor G specifically engages spontaneously achieved ribosome conformations while in an active, GTP-bound conformation to unlock and initiate peptidyl-tRNA translocation. These findings suggest that processes intrinsic to the pre-translocation ribosome complex can regulate the rate of protein synthesis, and that energy expenditure is used later in the translocation mechanism than previously proposed.
Asunto(s)
Factor G de Elongación Peptídica/metabolismo , Biosíntesis de Proteínas , Aminoacil-ARN de Transferencia/genética , Ribosomas/metabolismo , Codón , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , ARN Mensajero/genéticaRESUMEN
Bright, photostable, and nontoxic fluorescent contrast agents are critical for biological imaging. "Self-healing" dyes, in which triplet states are intramolecularly quenched, enable fluorescence imaging by increasing fluorophore brightness and longevity, while simultaneously reducing the generation of reactive oxygen species that promote phototoxicity. Here, we systematically examine the self-healing mechanism in cyanine-class organic fluorophores spanning the visible spectrum. We show that the Baird aromatic triplet-state energy of cyclooctatetraene can be physically altered to achieve order of magnitude enhancements in fluorophore brightness and signal-to-noise ratio in both the presence and absence of oxygen. We leverage these advances to achieve direct measurements of large-scale conformational dynamics within single molecules at submillisecond resolution using wide-field illumination and camera-based detection methods. These findings demonstrate the capacity to image functionally relevant conformational processes in biological systems in the kilohertz regime at physiological oxygen concentrations and shed important light on the multivariate parameters critical to self-healing organic fluorophore design.
Asunto(s)
Colorantes Fluorescentes/química , Línea Celular , Fluorescencia , Humanos , Microscopía FluorescenteRESUMEN
The substrate for ribosomes actively engaged in protein synthesis is a ternary complex of elongation factor Tu (EF-Tu), aminoacyl-tRNA (aa-tRNA), and GTP. EF-Tu plays a critical role in mRNA decoding by increasing the rate and fidelity of aa-tRNA selection at each mRNA codon. Here, using three-color single-molecule fluorescence resonance energy transfer imaging and molecular dynamics simulations, we examine the timing and role of conformational events that mediate the release of aa-tRNA from EF-Tu and EF-Tu from the ribosome after GTP hydrolysis. Our investigations reveal that conformational changes in EF-Tu coordinate the rate-limiting passage of aa-tRNA through the accommodation corridor en route to the peptidyl transferase center of the large ribosomal subunit. Experiments using distinct inhibitors of the accommodation process further show that aa-tRNA must at least partially transit the accommodation corridor for EF-Tuâ GDP to release. aa-tRNAs failing to undergo peptide bond formation at the end of accommodation corridor passage after EF-Tu release can be reengaged by EF-Tuâ GTP from solution, coupled to GTP hydrolysis. These observations suggest that additional rounds of ternary complex formation can occur on the ribosome during proofreading, particularly when peptide bond formation is slow, which may serve to increase both the rate and fidelity of protein synthesis at the expense of GTP hydrolysis.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Factor Tu de Elongación Peptídica/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Transferencia Resonante de Energía de Fluorescencia , Guanosina Trifosfato/metabolismo , Cinética , Factor Tu de Elongación Peptídica/genética , Biosíntesis de Proteínas , ARN de Transferencia/genética , Aminoacil-ARN de Transferencia/genética , Subunidades Ribosómicas Grandes/genética , Subunidades Ribosómicas Grandes/metabolismo , Ribosomas/genéticaRESUMEN
While surface-based single-molecule experiments have revolutionized our understanding of biology and biomolecules, the workflow in preparing for such experiments, especially surface cleaning and functionalization, remains labor-intensive and time-consuming. Even worse, meticulously assembled flow channels can be used only once for most experiments. A reusable surface would thus dramatically increase productivity and efficiency of single-molecule experiments. In this paper, we report a novel surface reconditioning strategy termed ERASE (Epitaxial Removal Aided by Strand Exchange) that allows a single flow cell to be used for vast repetition of single-molecule experiments. In this method, biomolecules immobilized to the surface through a nucleic acid duplex are liberated when a competing DNA strand disrupts the duplex via toehold-mediated strand displacement. We demonstrate the wide-range applicability of this method with various common surface preparation techniques, fluorescent dyes, and biomolecules including the bacterial ribosome. Beyond time and cost savings, we also show ERASE can assort molecules based on a nucleic acid barcode sequence, thus allowing experiments on different molecules in parallel. Our method increases the utility of prepared surfaces and is a significant improvement to the current single-use paradigm.
Asunto(s)
Oligodesoxirribonucleótidos/química , Carbocianinas , Colorantes Fluorescentes , Hibridación de Ácido Nucleico , Imagen Individual de MoléculaRESUMEN
Prevailing dogma holds that ribosomes are uniform in composition and function. Here, we show that nutrient limitation-induced stress in E. coli changes the relative expression of rDNA operons to alter the rRNA composition within the actively translating ribosome pool. The most upregulated operon encodes the unique 16S rRNA, rrsH, distinguished by conserved sequence variation within the small ribosomal subunit. rrsH-bearing ribosomes affect the expression of functionally coherent gene sets and alter the levels of the RpoS sigma factor, the master regulator of the general stress response. These impacts are associated with phenotypic changes in antibiotic sensitivity, biofilm formation, and cell motility and are regulated by stress response proteins, RelA and RelE, as well as the metabolic enzyme and virulence-associated protein, AdhE. These findings establish that endogenously encoded, naturally occurring rRNA sequence variation can modulate ribosome function, central aspects of gene expression regulation, and cellular physiology.
Asunto(s)
Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Modelos Moleculares , Operón , FenotipoRESUMEN
HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.
Asunto(s)
VIH-1/metabolismo , Conformación Proteica , Multimerización de Proteína , Productos del Gen env del Virus de la Inmunodeficiencia Humana/química , Antígenos CD4/química , Antígenos CD4/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Anticuerpos Anti-VIH/química , Anticuerpos Anti-VIH/metabolismo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/inmunología , Proteína gp120 de Envoltorio del VIH/metabolismo , Humanos , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Virión/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Aminoglycosides are chemically diverse, broad-spectrum antibiotics that target functional centers within the bacterial ribosome to impact all four principle stages (initiation, elongation, termination, and recycling) of the translation mechanism. The propensity of aminoglycosides to induce miscoding errors that suppress the termination of protein synthesis supports their potential as therapeutic interventions in human diseases associated with premature termination codons (PTCs). However, the sites of interaction of aminoglycosides with the eukaryotic ribosome and their modes of action in eukaryotic translation remain largely unexplored. Here, we use the combination of X-ray crystallography and single-molecule FRET analysis to reveal the interactions of distinct classes of aminoglycosides with the 80S eukaryotic ribosome. Crystal structures of the 80S ribosome in complex with paromomycin, geneticin (G418), gentamicin, and TC007, solved at 3.3- to 3.7-Å resolution, reveal multiple aminoglycoside-binding sites within the large and small subunits, wherein the 6'-hydroxyl substituent in ring I serves as a key determinant of binding to the canonical eukaryotic ribosomal decoding center. Multivalent binding interactions with the human ribosome are also evidenced through their capacity to affect large-scale conformational dynamics within the pretranslocation complex that contribute to multiple aspects of the translation mechanism. The distinct impacts of the aminoglycosides examined suggest that their chemical composition and distinct modes of interaction with the ribosome influence PTC read-through efficiency. These findings provide structural and functional insights into aminoglycoside-induced impacts on the eukaryotic ribosome and implicate pleiotropic mechanisms of action beyond decoding.
Asunto(s)
Aminoglicósidos/metabolismo , Eucariontes/efectos de los fármacos , Eucariontes/metabolismo , Ribosomas/metabolismo , Aminoglicósidos/química , Bacterias/genética , Bacterias/metabolismo , Sitios de Unión , Humanos , Modelos Moleculares , Conformación Molecular , Unión Proteica , Subunidades Ribosómicas/química , Subunidades Ribosómicas/metabolismo , Ribosomas/química , Ribosomas/genéticaRESUMEN
Phosphorylation-type (P-type) ATPases are ubiquitous primary transporters that pump cations across cell membranes through the formation and breakdown of a phosphoenzyme intermediate. Structural investigations suggest that the transport mechanism is defined by conformational changes in the cytoplasmic domains of the protein that are allosterically coupled to transmembrane helices so as to expose ion binding sites to alternate sides of the membrane. Here, we have used single-molecule fluorescence resonance energy transfer to directly observe conformational changes associated with the functional transitions in the Listeria monocytogenes Ca2+-ATPase (LMCA1), an orthologue of eukaryotic Ca2+-ATPases. We identify key intermediates with no known crystal structures and show that Ca2+ efflux by LMCA1 is rate-limited by phosphoenzyme formation. The transport process involves reversible steps and an irreversible step that follows release of ADP and extracellular release of Ca2+.
Asunto(s)
Adenosina Trifosfato/metabolismo , ATPasas Transportadoras de Calcio/química , ATPasas Transportadoras de Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Listeria monocytogenes/enzimología , Imagen Individual de Molécula , Adenosina Difosfato/metabolismo , Sitios de Unión , Calcio/metabolismo , Cinética , Modelos Moleculares , Fosforilación , Conformación ProteicaRESUMEN
The twister RNA is a recently discovered nucleolytic ribozyme that is present in both bacteria and eukarya. While its biological role remains unclear, crystal structure analyses and biochemical approaches have revealed critical features of its catalytic mechanism. Here, we set out to explore dynamic aspects of twister RNA folding along the cleavage reaction coordinate. To do so, we have employed both bulk and single-molecule fluorescence resonance energy transfer (FRET) methods to investigate a set of twister RNAs with labels strategically positioned at communicating segments. The data reveal that folding of the central pseudoknot (T1), the most crucial structural determinant to promote cleavage, exhibits reversible opening and closing dynamics at physiological Mg2+ concentration. Uncoupled folding, in which T1 formation precedes structuring for closing of stem P1, was confirmed using pre-steady-state three-color smFRET experiments initiated by Mg2+ injection. This finding suggests that the folding path of twister RNA requires proper orientation of the substrate prior to T1 closure such that the U5-A6 cleavage site becomes embraced to achieve its cleavage competent conformation. We also find that the cleaved 3'-fragment retains its compacted pseudoknot fold, despite the absence of the phylogenetically conserved stem P1, rationalizing the poor turnover efficiency of the twister ribozyme.
Asunto(s)
Transferencia Resonante de Energía de Fluorescencia , ARN Catalítico/metabolismo , Cinética , Modelos Moleculares , Pliegue del ARN , ARN Catalítico/químicaRESUMEN
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.
RESUMEN
Approximately 30% of the ATP generated in the living cell is utilized by P-type ATPase primary active transporters to generate and maintain electrochemical gradients across biological membranes. P-type ATPases undergo large conformational changes during their functional cycle to couple ATP hydrolysis in the cytoplasmic domains to ion transport across the membrane. The Ca(2+)-ATPase from Listeria monocytogenes, LMCA1, was found to be a suitable model of P-type ATPases and was engineered to facilitate single-molecule FRET studies of transport-related structural changes. Mutational analyses of the endogenous cysteine residues in LMCA1 were performed to reduce background labeling without compromising activity. Pairs of cysteines were introduced into the optimized low-reactivity background, and labeled with maleimide derivatives of Cy3 and Cy5 resulting in site-specifically double-labeled protein with moderate activity. Ensemble and confocal single-molecule FRET studies revealed changes in FRET distribution related to structural changes during the transport cycle, consistent with those observed by X-ray crystallography for the sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA). Notably, the cytosolic headpiece of LMCA1 was found to be distinctly more compact in the E1 state than in the E2 state. Thus, the established experimental system should allow future real-time FRET studies of the structural dynamics of LMCA1 as a representative P-type ATPase.
Asunto(s)
ATPasas Transportadoras de Calcio/genética , ATPasas Transportadoras de Calcio/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Listeria monocytogenes/enzimología , Ingeniería de Proteínas , ATPasas Transportadoras de Calcio/química , Maleimidas/química , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Conformación ProteicaRESUMEN
Mutations conferring resistance to translation inhibitors often alter the structure of rRNA. Reduced susceptibility to distinct structural antibiotic classes may, therefore, emerge when a common ribosomal binding site is perturbed, which significantly reduces the clinical utility of these agents. The translation inhibitors negamycin and tetracycline interfere with tRNA binding to the aminoacyl-tRNA site on the small 30S ribosomal subunit. However, two negamycin resistance mutations display unexpected differential antibiotic susceptibility profiles. Mutant U1060A in 16S Escherichia coli rRNA is resistant to both antibiotics, whereas mutant U1052G is simultaneously resistant to negamycin and hypersusceptible to tetracycline. Using a combination of microbiological, biochemical, single-molecule fluorescence transfer experiments, and X-ray crystallography, we define the specific structural defects in the U1052G mutant 70S E. coli ribosome that explain its divergent negamycin and tetracycline susceptibility profiles. Unexpectedly, the U1052G mutant ribosome possesses a second tetracycline binding site that correlates with its hypersusceptibility. The creation of a previously unidentified antibiotic binding site raises the prospect of identifying similar phenomena in antibiotic-resistant pathogens in the future.
Asunto(s)
Antibacterianos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ribosomas/genética , Tetraciclina/farmacología , Aminoácidos Diaminos/farmacología , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Mutación , ARN Bacteriano/genética , ARN Ribosómico/genéticaRESUMEN
Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Factor G de Elongación Peptídica/metabolismo , ARN Bacteriano/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/análisis , Transferencia Resonante de Energía de Fluorescencia , Guanosina Difosfato/análisis , Guanosina Difosfato/metabolismo , Modelos Moleculares , Factor G de Elongación Peptídica/análisis , Transporte de Proteínas , ARN Bacteriano/análisis , Aminoacil-ARN de Transferencia/análisis , Ribosomas/químicaRESUMEN
Single-molecule fluorescence microscopy is uniquely suited for detecting transient molecular recognition events, yet achieving the time resolution and statistics needed to realize this potential has proven challenging. Here we present a single-molecule imaging and analysis platform using scientific complementary metal-oxide semiconductor (sCMOS) detectors that enables imaging of 15,000 individual molecules simultaneously at millisecond rates. This system enabled the detection of previously obscured processes relevant to the fidelity mechanism in protein synthesis.
Asunto(s)
Procesamiento de Imagen Asistido por Computador/métodos , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/métodos , Imagen Molecular/métodos , ARN de Transferencia/ultraestructura , Ribosomas/ultraestructura , Algoritmos , Bacterias/ultraestructura , Transferencia Resonante de Energía de Fluorescencia , Humanos , Imagen Molecular/instrumentación , Factores de TiempoRESUMEN
Bright, long-lasting and non-phototoxic organic fluorophores are essential to the continued advancement of biological imaging. Traditional approaches towards achieving photostability, such as the removal of molecular oxygen and the use of small-molecule additives in solution, suffer from potentially toxic side effects, particularly in the context of living cells. The direct conjugation of small-molecule triplet state quenchers, such as cyclooctatetraene (COT), to organic fluorophores has the potential to bypass these issues by restoring reactive fluorophore triplet states to the ground state through intra-molecular triplet energy transfer. Such methods have enabled marked improvement in cyanine fluorophore photostability spanning the visible spectrum. However, the generality of this strategy to chemically and structurally diverse fluorophore species has yet to be examined. Here, we show that the proximal linkage of COT increases the photon yield of a diverse range of organic fluorophores widely used in biological imaging applications, demonstrating that the intra-molecular triplet energy transfer mechanism is a potentially general approach for improving organic fluorophore performance and photostability.