scholarly journals Guiding biomolecular interactions in cells using de novo protein-protein interfaces

2018 ◽  
Author(s):  
Abigail J Smith ◽  
Franziska Thomas ◽  
Deborah Shoemark ◽  
Derek N Woolfson ◽  
Nigel J Savery
Keyword(s):  
Rational Design ◽  
De Novo ◽  
Gene Activation ◽  
Low Complexity ◽  
Coiled Coils ◽  
Biomolecular Interactions ◽  
Biomolecular Systems ◽  
Binding Domains ◽  
In Cells

An improved ability to direct and control biomolecular interactions in living cells would impact on synthetic biology. A key issue is the need to introduce interacting components that act orthogonally to endogenous proteomes and interactomes. Here we show that low-complexity, de novo designed protein-protein-interaction (PPI) domains can substitute for natural PPIs and guide engineered protein-DNA interactions in Escherichia coli. Specifically, we use de novo homo- and hetero-dimeric coiled coils to reconstitute a cytoplasmic split adenylate cyclase; to recruit RNA polymerase to a promoter and activate gene expression; and to oligomerize both natural and designed DNA-binding domains to repress transcription. Moreover, the stabilities of the heterodimeric coiled coils can be modulated by rational design and, thus, adjust the levels of gene activation and repression in vivo. These experiments demonstrate the possibilities for using designed proteins and interactions to control biomolecular systems such as enzyme cascades and circuits in cells.

scholarly journals Tuning levels of low-complexity domain interactions to modulate endogenous oncogenic transcription

2021 ◽  
Author(s):  
Shasha Chong ◽  
Thomas G. W. Graham ◽  
Claire Dugast-Darzacq ◽  
Gina M. Dailey ◽  
Xavier Darzacq ◽  
...  
Keyword(s):  
Single Molecule ◽  
Target Genes ◽  
Gene Activation ◽  
Low Complexity ◽  
Intrinsically Disordered ◽  
Human Genes ◽  
Domain Interactions ◽  
Bona Fide ◽  
Liquid Liquid Phase Separation

Gene activation by mammalian transcription factors (TFs) requires dynamic, multivalent, and selective interactions of their intrinsically disordered low-complexity domains (LCDs), but how such interactions mediate transcription remains unclear. It has been proposed that extensive LCD-LCD interactions culminating in liquid-liquid phase separation (LLPS) of TFs is the dominant mechanism underlying transactivation. Here, we investigated how tuning the amount and localization of LCD-LCD interactions in vivo affects transcription of endogenous human genes. Quantitative single-cell and single-molecule imaging reveals that the oncogenic TF EWS/FLI1 requires a finely tuned range of LCD-LCD interactions to efficiently activate target genes. Modest or more dramatic increases in LCD-LCD interactions toward putative LLPS repress EWS/FLI1-driven transcription in patient cells. Likewise, ectopically creating LCD-LCD interactions to sequester EWS/FLI1 into a bona fide LLPS compartment, the nucleolus, inhibits EWS/FLI1-driven transcription and oncogenic transformation. Our findings reveal fundamental principles underlying LCD-mediated transcription and suggest mislocalizing specific LCD-LCD interactions as a novel therapeutic strategy for targeting disease-causing TFs.

scholarly journals Unique Posttranslational Modifications of Chitin-Binding Lectins of Entamoeba invadens Cyst Walls

2006 ◽  
Vol 5 (5) ◽  
pp. 836-848 ◽  
Author(s):  
Katrina L. Van Dellen ◽  
Anirban Chatterjee ◽  
Daniel M. Ratner ◽  
Paula E. Magnelli ◽  
John F. Cipollo ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Cyst Wall ◽  
Low Complexity ◽  
Pcr Analysis ◽  
Major Protein ◽  
Chitin Binding Domain ◽  
Binding Domains ◽  
Reverse Transcription Pcr ◽  
Entamoeba Invadens

ABSTRACT Entamoeba histolytica, which causes amebic dysentery and liver abscesses, is spread via chitin-walled cysts. The most abundant protein in the cyst wall of Entamoeba invadens, a model for amebic encystation, is a lectin called EiJacob1. EiJacob1 has five tandemly arrayed, six-Cys chitin-binding domains separated by low-complexity Ser- and Thr-rich spacers. E. histolytica also has numerous predicted Jessie lectins and chitinases, which contain a single, N-terminal eight-Cys chitin-binding domain. We hypothesized that E. invadens cyst walls are composed entirely of proteins with six-Cys or eight-Cys chitin-binding domains and that some of these proteins contain sugars. E. invadens genomic sequences predicted seven Jacob lectins, five Jessie lectins, and three chitinases. Reverse transcription-PCR analysis showed that mRNAs encoding Jacobs, Jessies, and chitinases are increased during E. invadens encystation, while mass spectrometry showed that the cyst wall is composed of an ∼30:70 mix of Jacob lectins (cross-linking proteins) and Jessie and chitinase lectins (possible enzymes). Three Jacob lectins were cleaved prior to Lys at conserved sites (e.g., TPSVDK) in the Ser- and Thr-rich spacers between chitin-binding domains. A model peptide was cleaved at the same site by papain and E. invadens Cys proteases, suggesting that the latter cleave Jacob lectins in vivo. Some Jacob lectins had O-phosphodiester-linked carbohydrates, which were one to seven hexoses long and had deoxysugars at reducing ends. We concluded that the major protein components of the E. invadens cyst wall all contain chitin-binding domains (chitinases, Jessie lectins, and Jacob lectins) and that the Jacob lectins are differentially modified by site-specific Cys proteases and O-phosphodiester-linked glycans.

scholarly journals Direct single-molecule measurements of phycocyanobilin photophysics in monomeric C-phycocyanin

2017 ◽  
Vol 114 (37) ◽  
pp. 9779-9784 ◽  
Author(s):  
Allison H. Squires ◽  
W. E. Moerner
Keyword(s):  
Energy Transfer ◽  
Single Molecule ◽  
Rational Design ◽  
Light Adaptation ◽  
De Novo ◽  
Photosynthetic Apparatus ◽  
Emission Spectra ◽  
Free Solution ◽  
Surface Attachment

Phycobilisomes are highly organized pigment–protein antenna complexes found in the photosynthetic apparatus of cyanobacteria and rhodophyta that harvest solar energy and transport it to the reaction center. A detailed bottom-up model of pigment organization and energy transfer in phycobilisomes is essential to understanding photosynthesis in these organisms and informing rational design of artificial light-harvesting systems. In particular, heterogeneous photophysical behaviors of these proteins, which cannot be predicted de novo, may play an essential role in rapid light adaptation and photoprotection. Furthermore, the delicate architecture of these pigment–protein scaffolds sensitizes them to external perturbations, for example, surface attachment, which can be avoided by study in free solution or in vivo. Here, we present single-molecule characterization of C-phycocyanin (C-PC), a three-pigment biliprotein that self-assembles to form the midantenna rods of cyanobacterial phycobilisomes. Using the Anti-Brownian Electrokinetic (ABEL) trap to counteract Brownian motion of single particles in real time, we directly monitor the changing photophysical states of individual C-PC monomers from Spirulina platensis in free solution by simultaneous readout of their brightness, fluorescence anisotropy, fluorescence lifetime, and emission spectra. These include single-chromophore emission states for each of the three covalently bound phycocyanobilins, providing direct measurements of the spectra and photophysics of these chemically identical molecules in their native protein environment. We further show that a simple Förster resonant energy transfer (FRET) network model accurately predicts the observed photophysical states of C-PC and suggests highly variable quenching behavior of one of the chromophores, which should inform future studies of higher-order complexes.

scholarly journals Correction: A ratiometric fluorescent probe for peroxynitrite prepared by de novo synthesis and its application in assessing the mitochondrial oxidative stress status in cells and in vivo

2018 ◽  
Vol 54 (93) ◽  
pp. 13159-13159 ◽  
Author(s):  
Dong-Ye Zhou ◽  
Yongfei Li ◽  
Wen-Li Jiang ◽  
Yang Tian ◽  
Junjie Fei ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Fluorescent Probe ◽  
De Novo ◽  
De Novo Synthesis ◽  
Mitochondrial Oxidative Stress ◽  
Oxidative Stress Status ◽  
Ratiometric Fluorescent Probe ◽  
In Cells

Correction for ‘A ratiometric fluorescent probe for peroxynitrite prepared by de novo synthesis and its application in assessing the mitochondrial oxidative stress status in cells and in vivo’ by Dong-Ye Zhou et al., Chem. Commun., 2018, 54, 11590–11593.

scholarly journals Comparison of In Vivo Selection and Rational Design of Heterodimeric Coiled Coils

Structure ◽  
2002 ◽  
Vol 10 (9) ◽  
pp. 1235-1248 ◽  
Author(s):  
Katja M. Arndt ◽  
Joelle N. Pelletier ◽  
Kristian M. Müller ◽  
Andreas Plückthun ◽  
Tom Alber
Keyword(s):  
Rational Design ◽  
Coiled Coils ◽  
In Vivo Selection

scholarly journals Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

1998 ◽  
Vol 9 (5) ◽  
pp. 1007-1023 ◽  
Author(s):  
Peter Lorenz ◽  
Brenda F. Baker ◽  
C. Frank Bennett ◽  
David L. Spector
Keyword(s):  
Antisense Oligonucleotides ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Nuclear Body ◽  
Nuclear Bodies ◽  
Phosphorothioate Oligodeoxynucleotides ◽  
Nuclear Structures ◽  
In Cells

Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), which were set against a diffuse nucleoplasmic population excluding nucleoli. PS bodies are nuclear structures that formed in cells after PS-ON delivery by transfection agents or microinjection but were observed irrespectively of antisense activity or sequence. Ultrastructurally, PS bodies corresponded to electron-dense structures of 150–300 nm diameter and resembled nuclear bodies that were found with lower frequency in cells lacking PS-ONs. The environment of a living cell was required for the de novo formation of PS bodies, which occurred within minutes after the introduction of PS-ONs. PS bodies were stable entities that underwent noticeable reorganization only during mitosis. Upon exit from mitosis, PS bodies were assembled de novo from diffuse PS-ON pools in the daughter nuclei. In situ fractionation demonstrated an association of PS-ONs with the nuclear matrix. Taken together, our data provide evidence for the formation of a nuclear body in cells after introduction of phosphorothioate oligodeoxynucleotides.

scholarly journals Computational Approaches to Investigate and Design Lipid-binding Domains for Membrane Biosensing

2021 ◽  
Vol 75 (12) ◽  
pp. 1031-1036
Author(s):  
Sriraksha Srinivasan ◽  
Stefano Vanni
Keyword(s):  
Membrane Trafficking ◽  
De Novo ◽  
Md Simulations ◽  
Lipid Binding ◽  
Broad Perspective ◽  
Computational Approaches ◽  
Binding Domains ◽  
Specific Lipid

Association of proteins with cellular membranes is critical for signaling and membrane trafficking processes. Many peripheral lipid-binding domains have been identified in the last few decades and have been investigated for their specific lipid-sensing properties using traditional in vivo and in vitro studies. However, several knowledge gaps remain owing to intrinsic limitations of these methodologies. Thus, novel approaches are necessary to further our understanding in lipid–protein biology. This review briefly discusses lipid-binding domains that act as specific lipid biosensors and provides a broad perspective on the computational approaches such as molecular dynamics (MD) simulations and machine learning (ML)-based techniques that can be used to study protein–membrane interactions. We also highlight the need for de novo design of proteins that elicit specific lipid-binding properties.

scholarly journals TriPepSVM - de novo prediction of RNA-binding proteins based on short amino acid motifs

2018 ◽  
Author(s):  
Annkatrin Bressin ◽  
Roman Schulte-Sasse ◽  
Davide Figini ◽  
Erika C Urdaneta ◽  
Benedikt M Beckmann ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Rna Binding ◽  
De Novo ◽  
Rna Binding Proteins ◽  
Low Complexity ◽  
Structural Features ◽  
Support Vector ◽  
String Kernels ◽  
Rna Binders

In recent years hundreds of novel RNA-binding proteins (RBPs) have been identified leading to the discovery of novel RNA-binding domains (RBDs). Furthermore, unstructured or disordered low-complexity regions of RBPs have been identified to play an important role in interactions with nucleic acids. However, these advances in understanding RBPs are limited mainly to eukaryotic species and we only have limited tools to faithfully predict RNA-binders from bacteria. Here, we describe a support vector machine (SVM)-based method, called TriPepSVM, for the classification of RNA-binding proteins and non-RBPs. TriPepSVM applies string kernels to directly handle protein sequences using tri-peptide frequencies. Testing the method in human and bacteria, we find that several RBP-enriched tripeptides occur more often in structurally disordered regions of RBPs. TriPepSVM outperforms existing applications, which consider classical structural features of RNA-binding or homology, in the task of RBP prediction in both human and bacteria. Finally, we predict 66 novel RBPs in Salmonella Typhimurium and validate the bacterial proteins ClpX, DnaJ and UbiG to associate with RNA in vivo.

scholarly journals A convolutional neural network for the prediction and forward design of ribozyme-based gene-control elements

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Calvin M Schmidt ◽  
Christina D Smolke
Keyword(s):  
Machine Learning ◽  
Rational Design ◽  
De Novo ◽  
Regulation Of Gene Expression ◽  
Regulatory Activity ◽  
Rna Sequences ◽  
Genetic Switch ◽  
Gene Regulatory ◽  
Functional Rnas

Ribozyme switches are a class of RNA-encoded genetic switch that support conditional regulation of gene expression across diverse organisms. An improved elucidation of the relationships between sequence, structure, and activity can improve our capacity for de novo rational design of ribozyme switches. Here, we generated data on the activity of hundreds of thousands of ribozyme sequences. Using automated structural analysis and machine learning, we leveraged these large datasets to develop predictive models that estimate the in vivo gene-regulatory activity of a ribozyme sequence. These models supported the de novo design of ribozyme libraries with low mean basal gene-regulatory activities and new ribozyme switches that exhibit changes in gene-regulatory activity in the presence of a target ligand, producing functional switches for four out of five aptamers. Our work examines how biases in the model and the dataset that affect prediction accuracy can arise and demonstrates that machine learning can be applied to RNA sequences to predict gene-regulatory activity, providing the basis for design tools for functional RNAs.

Sign in / Sign up

Export Citation Format

Share Document