scholarly journals Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

PLoS ONE ◽  
2020 ◽  
Vol 15 (11) ◽  
pp. e0242592
Author(s):  
Shuqin Zhou ◽  
Siyu Feng ◽  
David Brown ◽  
Bo Huang
Keyword(s):  
Directed Evolution ◽  
Signal Amplification ◽  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Protein Labeling ◽  
Small Fragment ◽  
Wide Range ◽  
Split System ◽  
Low Expression

The flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

scholarly journals Improved yellow-green split fluorescent proteins for protein labeling and signal amplification

2020 ◽  
Author(s):  
Shuqin Zhou ◽  
Siyu Feng ◽  
David Brown ◽  
Bo Huang
Keyword(s):  
Directed Evolution ◽  
Signal Amplification ◽  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Protein Labeling ◽  
Small Fragment ◽  
Wide Range ◽  
Split System ◽  
Low Expression

AbstractThe flexibility and versatility of self-complementing split fluorescent proteins (FPs) have enabled a wide range of applications. In particular, the FP1-10/11 split system contains a small fragment that facilitates efficient generation of endogenous-tagged cell lines and animals as well as signal amplification using tandem FP11 tags. To improve the FP1-10/11 toolbox we previously developed, here we used a combination of directed evolution and rational design approaches, resulting in two mNeonGreen (mNG)-based split FPs (mNG3A1-10/11 and mNG3K1-10/11) and one mClover-based split FP (CloGFP1-10/11). mNG3A1-10/11 and mNG3K1-10/11 not only enhanced the complementation efficiency at low expression levels, but also allowed us to demonstrate signal amplification using tandem mNG211 fragments in mammalian cells.

Directed Evolution of Protein Catalysts

2018 ◽  
Vol 87 (1) ◽  
pp. 131-157 ◽  
Author(s):  
Cathleen Zeymer ◽  
Donald Hilvert
Keyword(s):  
Directed Evolution ◽  
Rational Design ◽  
Chemical Transformations ◽  
Catalytic Promiscuity ◽  
Catalytic Activities ◽  
Effective Strategies ◽  
Wide Range ◽  
Protein Properties ◽  
New Protein ◽  
Modify Protein

Directed evolution is a powerful technique for generating tailor-made enzymes for a wide range of biocatalytic applications. Following the principles of natural evolution, iterative cycles of mutagenesis and screening or selection are applied to modify protein properties, enhance catalytic activities, or develop completely new protein catalysts for non-natural chemical transformations. This review briefly surveys the experimental methods used to generate genetic diversity and screen or select for improved enzyme variants. Emphasis is placed on a key challenge, namely how to generate novel catalytic activities that expand the scope of natural reactions. Two particularly effective strategies, exploiting catalytic promiscuity and rational design, are illustrated by representative examples of successfully evolved enzymes. Opportunities for extending these approaches to more complex biocatalytic systems are also considered.

scholarly journals LSSmScarlet, dCyRFP2s, dCyOFP2s and CRISPRed2s, Genetically Encoded Red Fluorescent Proteins with a Large Stokes Shift

2021 ◽  
Vol 22 (23) ◽  
pp. 12887
Author(s):  
Oksana M. Subach ◽  
Anna V. Vlaskina ◽  
Yuliya K. Agapova ◽  
Pavel V. Dorovatovskii ◽  
Alena Y. Nikolaeva ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Rational Design ◽  
Fluorescent Proteins ◽  
Stokes Shift ◽  
Confocal Imaging ◽  
Large Stokes Shift ◽  
Two Photon ◽  
Dual Color ◽  
Red Fluorescent Proteins

Genetically encoded red fluorescent proteins with a large Stokes shift (LSSRFPs) can be efficiently co-excited with common green FPs both under single- and two-photon microscopy, thus enabling dual-color imaging using a single laser. Recent progress in protein development resulted in a great variety of novel LSSRFPs; however, the selection of the right LSSRFP for a given application is hampered by the lack of a side-by-side comparison of the LSSRFPs’ performance. In this study, we employed rational design and random mutagenesis to convert conventional bright RFP mScarlet into LSSRFP, called LSSmScarlet, characterized by excitation/emission maxima at 470/598 nm. In addition, we utilized the previously reported LSSRFPs mCyRFP1, CyOFP1, and mCRISPRed as templates for directed molecular evolution to develop their optimized versions, called dCyRFP2s, dCyOFP2s and CRISPRed2s. We performed a quantitative assessment of the developed LSSRFPs and their precursors in vitro on purified proteins and compared their brightness at 488 nm excitation in the mammalian cells. The monomeric LSSmScarlet protein was successfully utilized for the confocal imaging of the structural proteins in live mammalian cells and multicolor confocal imaging in conjugation with other FPs. LSSmScarlet was successfully applied for dual-color two-photon imaging in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet protein at the resolution of 1.4 Å that revealed a hydrogen bond network supporting excited-state proton transfer (ESPT). Quantum mechanics/molecular mechanics molecular dynamic simulations confirmed the ESPT mechanism of a large Stokes shift. Structure-guided mutagenesis revealed the role of R198 residue in ESPT that allowed us to generate a variant with improved pH stability. Finally, we showed that LSSmScarlet protein is not appropriate for STED microscopy as a consequence of LSSRed-to-Red photoconversion with high-power 775 nm depletion light.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

Evaluation of the nucleopolyhedrovirus of Anticarsia gemmatalis as a vector for gene therapy in mammals

2020 ◽  
Vol 20 ◽  
Author(s):  
Cintia N. Parsza ◽  
Diego L. Mengual Gómez ◽  
Jorge Alejandro Simonin ◽  
Mariano Nicolás Belaich ◽  
Pablo Daniel Ghiringhelli
Keyword(s):  
Gene Therapy ◽  
Mammalian Cells ◽  
Insect Cells ◽  
Autographa Californica ◽  
Anticarsia Gemmatalis ◽  
Agricultural Pests ◽  
Mammalian Cell Lines ◽  
Delivery Vector ◽  
Wide Range ◽  
Insect Pathogens

Background: Baculoviruses are insect pathogens with important biotechnological applications that transcend their use as biological controllers of agricultural pests. One species, Autographa californica multiple nucleopolhyedrovirus (AcMNPV) has been extensively exploited as a molecular platform to produce recombinant proteins and as a delivery vector for genes in mammals, because it can transduce a wide range of mammalian cells and tissues without replicating or producing progeny. Objective/Method: To investigate if the budded virions of Anticarsia gemmatalis multiple nucleopolhyedrovirus (AgMNPV) species has the same ability, the viral genome was modified by homologous recombination into susceptible insect cells to integrate reporter genes and then it was evaluated on mammalian cell lines in comparative form with respect to equivalent viruses derived from AcMNPV. Besides, the replicative capacity of AgMNPV´s virions in mammals was determined. Results: The experiments carried out showed that the recombinant variant of AgMNPV transduces and support the expression of delivered genes but not replicates in mammalian cells. Conclusion: Consequently, this insect pathogen is proposed as an alternative of non-infectious viruses in humans to explore new approaches in gene therapy and other applications based on the use of mammalian cells.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals Fluorescent thermal shift-based method for detection of NF-κB binding to double-stranded DNA

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter D. Leitner ◽  
Ilja Vietor ◽  
Lukas A. Huber ◽  
Taras Valovka
Keyword(s):  
Dna Binding ◽  
Rational Design ◽  
Dissociation Constants ◽  
Inflammatory Diseases ◽  
Dimethyl Fumarate ◽  
Bioinformatic Analysis ◽  
Quantitative Detection ◽  
Regulatory Sequences ◽  
Wide Range ◽  
Inhibitory Drug

AbstractThe nuclear factor kappa B (NF-κB) family of dimeric transcription factors regulates a wide range of genes by binding to their specific DNA regulatory sequences. NF-κB is an important therapeutic target linked to a number of cancers as well as autoimmune and inflammatory diseases. Therefore, effective high-throughput methods for the detection of NF-κB DNA binding are essential for studying its transcriptional activity and for inhibitory drug screening. We describe here a novel fluorescence-based assay for quantitative detection of κB consensus double-stranded (ds) DNA binding by measuring the thermal stability of the NF-κB proteins. Specifically, DNA binding proficient NF-κB probes, consisting of the N-terminal p65/RelA (aa 1–306) and p50 (aa 1–367) regions, were designed using bioinformatic analysis of protein hydrophobicity, folding and sequence similarities. By measuring the SYPRO Orange fluorescence during thermal denaturation of the probes, we detected and quantified a shift in the melting temperatures (ΔTm) of p65/RelA and p50 produced by the dsDNA binding. The increase in Tm was proportional to the concentration of dsDNA with apparent dissociation constants (KD) of 2.228 × 10–6 M and 0.794 × 10–6 M, respectively. The use of withaferin A (WFA), dimethyl fumarate (DMF) and p-xyleneselenocyanate (p-XSC) verified the suitability of this assay for measuring dose-dependent antagonistic effects on DNA binding. In addition, the assay can be used to analyse the direct binding of inhibitors and their effects on structural stability of the protein probe. This may facilitate the identification and rational design of new drug candidates interfering with NF-κB functions.

Experiences with alluvial foundations for earth dams in the Prairie provinces

1979 ◽  
Vol 16 (2) ◽  
pp. 255-271 ◽  
Author(s):  
N. Peters ◽  
K. N. Lamb
Keyword(s):  
Rational Design ◽  
Preliminary Design ◽  
Empirical Relationships ◽  
Design Concepts ◽  
Theoretical Design ◽  
Pore Pressures ◽  
Wide Range ◽  
Thick Layers ◽  
Limited Testing ◽  
Prairie Provinces

The foundations for numerous dams in proglacial and interglacial valleys in the Prairie provinces consist of soft alluvial soils. The deposits are up to 60 m deep, and contain thick layers of clay interspersed with lenses and layers of silt, sand, and gravel.This paper describes the damsite investigation and laboratory testing required, the design methods and construction procedures used, and the foundation performance observed during and after construction. A number of empirical relationships between index tests and physical properties of the soils, which provide useful guidelines for preliminary design, are presented.The design approach has gradually evolved from an empirical design with limited testing to a more rational design based on detailed investigations and thorough instrumentation. Increased reliance is placed on observational apparatus to monitor movements and pore pressures to confirm design assumptions as construction proceeds. The theoretical design is always checked with former designs of dams that have performed satisfactorily.Safe economical dams have been constructed in spite of large deformations and high pore pressures. Two case histories illustrate the wide range in dam design for alluvial foundations. The first shows an older design cross section with modifications required to ensure a stable dam, and the second describes a recently constructed dam that incorporates many of the latest design concepts.

scholarly journals A novel human Smad4 mutation is involved in papillary thyroid carcinoma progression

2011 ◽  
Vol 19 (1) ◽  
pp. 39-55 ◽  
Author(s):  
Sonia D'Inzeo ◽  
Arianna Nicolussi ◽  
Caterina Francesca Donini ◽  
Massimo Zani ◽  
Patrizia Mancini ◽  
...  
Keyword(s):  
Papillary Thyroid Carcinoma ◽  
Thyroid Carcinoma ◽  
Mammalian Cells ◽  
Transforming Growth Factor ◽  
Human Cancer ◽  
Papillary Thyroid ◽  
Tgfβ Signaling ◽  
Strong Reduction ◽  
Mesenchymal Transition ◽  
Wide Range

Smad proteins are the key effectors of the transforming growth factor β (TGFβ) signaling pathway in mammalian cells. Smad4 plays an important role in human physiology, and its mutations were found with high frequency in wide range of human cancer. In this study, we have functionally characterized Smad4 C324Y mutation, isolated from a nodal metastasis of papillary thyroid carcinoma. We demonstrated that the stable expression of Smad4 C324Y in FRTL-5 cells caused a significant activation of TGFβ signaling, responsible for the acquisition of transformed phenotype and invasive behavior. The coexpression of Smad4 C324Y with Smad4 wild-type determined an increase of homo-oligomerization of Smad4 with receptor-regulated Smads and a lengthening of nuclear localization. FRTL-5 clones overexpressing Smad4 C324Y showed a strong reduction of response to antiproliferative action of TGFβ1, acquired the ability to grow in anchorage-independent conditions, showed a fibroblast-like appearance and a strong reduction of the level of E-cadherin, one crucial event of the epithelial–mesenchymal transition process. The acquisition of a mesenchymal phenotype gave the characteristics of increased cellular motility and a significant reduction in adhesion to substrates such as fibronectin and laminin. Overall, our results demonstrate that the Smad4 C324Y mutation plays an important role in thyroid carcinogenesis and can be considered as a new prognostic and therapeutic target for thyroid cancer.

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