18 research outputs found

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

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    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

    Engineering native and artificial heme c containing proteins for biochemical applications and studies of protein folding

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    Thesis (Ph. D.)--University of Rochester. Dept. of Chemistry, 2012.Heme c containing proteins are known for their intense colors and essential functions in nature. These proteins contain heme that is covalently bound to the protein. For part of the work in this thesis, we have developed fusion tags that contain heme c, known as heme-tags, that reversibly bind an L-histidine immobilized Sepharose (HIS) chromatography resin for affinity purification of recombinant proteins expressed in Escherichia coli. The heme-tag HIS purification method couples the ease of affinity purification with the convenience of visible detection for protein tracking. In addition, we show that the heme-tag can be used to quantify the protein. Heme is covalently bound to native heme c proteins by several dedicated biogenesis systems that exhibit a variable degree of diversity and substrate specificity. Heme-tagged proteins are produced using a biogenesis system native to E. coli with promiscuous substrate specificity. The biogenesis system that matures native mitochondrial cytochromes c (cyt c) that function in cellular respiration and apoptosis is referred to as cytochrome c heme lyase (CCHL). The substrate specificity of CCHL has been explored, but the details are unclear. In this thesis, we show that CCHL can mature the first 18 N-terminal residues of horse cyt c fused to a non-heme containing protein, which demonstrates that the C-terminal portion of cyt c is unimportant for substrate recognition. This work lends new insight into the substrate specificity of CCHL and provides a new approach for producing artificial heme c proteins that could find use in multiple applications. In addition to developing novel biochemical applications of the heme tag, we study the folding of the important model protein horse cyt c. Protein folding is considered one of the most confounding problems in science, and cyt c has long been a model protein to understand the general principles of folding. Herein, we show that the folding of cyt c is more complex than previously reported using single molecule fluorescence methods. We show evidence of multiple folding intermediates and pathways as the protein folds on its energy landscape

    Efficient and Flexible Preparation of Biosynthetic Microperoxidases

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    Heme peptides and their derivatives, also called microperoxidases (MPs), are employed as heme protein active site models, catalysts, and charge-transfer chromophores. In this work, two approaches to the biosynthesis of novel MPs are described. In one method, heme peptides are expressed as C-terminal tags to the protein azurin and the MP is liberated by proteolytic cleavage by an endopeptidase. In an alternative approach, heme peptides are expressed as N-terminal tags to the cysteine protease domain (CPD) of the <i>Vibrio cholerae</i> MARTX toxin. Once activated by inositol hexakisphosphate, CPD undergoes autocleavage at an N-terminal leucine residue to liberate the MP. Purification is aided by use of a histidine-immobilized Sepharose column that binds exposed heme [Asher, W. A., and Bren, K. L. (2010) <i>Protein Sci. 19</i>, 1830–1839]. These methods provide efficient and adaptable routes to the preparation of a wide range of biosynthetic heme peptides

    Single-Molecule Analysis of Cytochrome <i>c</i> Folding by Monitoring the Lifetime of an Attached Fluorescent Probe

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    Conformational dynamics of proteins are important for function. However, obtaining information about specific conformations is difficult for samples displaying heterogeneity. Here, time-resolved fluorescence resonance energy transfer is used to characterize the folding of single cytochrome <i>c</i> molecules. In particular, measurements of the fluorescence lifetimes of individual cytochrome <i>c</i> molecules labeled with a single dye that is quenched by energy transfer to the heme were used to monitor conformational transitions of the protein under partially denaturing conditions. These studies indicate significantly more conformational heterogeneity than has been described previously. Importantly, the use of a purified singly labeled sample made a direct comparison to ensemble data possible. The distribution of lifetimes of single proteins was compared to the distribution of lifetimes determined from analysis of ensemble lifetime fluorescence data. The results show broad agreement between single-molecule and ensemble data, with a similar range of lifetimes. However, the single-molecule data reveal greater conformational heterogeneity

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

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    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 approach and its application to the gamut of fluorophore species commonly used for biomedical imaging

    Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

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    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

    Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates.

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    Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps \u3e2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line-derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for \u3e50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, an
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