scholarly journals Nucleosomes accelerate transcription factor dissociation

2013 ◽  
Vol 42 (5) ◽  
pp. 3017-3027 ◽  
Author(s):  
Yi Luo ◽  
Justin A. North ◽  
Sean D. Rose ◽  
Michael G. Poirier
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
High Specificity ◽  
Dissociation Rate ◽  
Duplex Dna ◽  
Recognition Sequence ◽  
Nucleosome Arrays

AbstractTranscription factors (TF) bind DNA-target sites within promoters to activate gene expression. TFs target their DNA-recognition sequences with high specificity by binding with resident times of up to hours in vitro. However, in vivo TFs can exchange on the order of seconds. The factors that regulate TF dynamics in vivo and increase dissociation rates by orders of magnitude are not known. We investigated TF binding and dissociation dynamics at their recognition sequence within duplex DNA, single nucleosomes and short nucleosome arrays with single molecule total internal reflection fluorescence (smTIRF) microscopy. We find that the rate of TF dissociation from its site within either nucleosomes or nucleosome arrays is increased by 1000-fold relative to duplex DNA. Our results suggest that TF binding within chromatin could be responsible for the dramatic increase in TF exchange in vivo. Furthermore, these studies demonstrate that nucleosomes regulate DNA–protein interactions not only by preventing DNA–protein binding but by dramatically increasing the dissociation rate of protein complexes from their DNA-binding sites.

scholarly journals Interfacial Peptides as Affinity Modulating Agents of Protein-Protein Interactions

Biomolecules ◽  
2022 ◽  
Vol 12 (1) ◽  
pp. 106
Author(s):  
Pavel V. Ershov ◽  
Yuri V. Mezentsev ◽  
Alexis S. Ivanov
Keyword(s):  
Protein Interactions ◽  
Targeted Delivery ◽  
Protein Complexes ◽  
Protein Protein Interactions ◽  
Good Efficiency ◽  
Cellular Targets ◽  
Proteolytic Stability ◽  
Clinically Significant

The identification of disease-related protein-protein interactions (PPIs) creates objective conditions for their pharmacological modulation. The contact area (interfaces) of the vast majority of PPIs has some features, such as geometrical and biochemical complementarities, “hot spots”, as well as an extremely low mutation rate that give us key knowledge to influence these PPIs. Exogenous regulation of PPIs is aimed at both inhibiting the assembly and/or destabilization of protein complexes. Often, the design of such modulators is associated with some specific problems in targeted delivery, cell penetration and proteolytic stability, as well as selective binding to cellular targets. Recent progress in interfacial peptide design has been achieved in solving all these difficulties and has provided a good efficiency in preclinical models (in vitro and in vivo). The most promising peptide-containing therapeutic formulations are under investigation in clinical trials. In this review, we update the current state-of-the-art in the field of interfacial peptides as potent modulators of a number of disease-related PPIs. Over the past years, the scientific interest has been focused on following clinically significant heterodimeric PPIs MDM2/p53, PD-1/PD-L1, HIF/HIF, NRF2/KEAP1, RbAp48/MTA1, HSP90/CDC37, BIRC5/CRM1, BIRC5/XIAP, YAP/TAZ–TEAD, TWEAK/FN14, Bcl-2/Bax, YY1/AKT, CD40/CD40L and MINT2/APP.

scholarly journals Evolutionary diversification of protein–protein interactions by interface add-ons

2017 ◽  
Vol 114 (40) ◽  
pp. E8333-E8342 ◽  
Author(s):  
Maximilian G. Plach ◽  
Florian Semmelmann ◽  
Florian Busch ◽  
Markus Busch ◽  
Leonhard Heizinger ◽  
...  
Keyword(s):  
Protein Interaction ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Evolutionary Strategy ◽  
Structural Level ◽  
Protein Protein Interactions ◽  
Protein Protein Interaction ◽  
Evolutionary Diversification

Cells contain a multitude of protein complexes whose subunits interact with high specificity. However, the number of different protein folds and interface geometries found in nature is limited. This raises the question of how protein–protein interaction specificity is achieved on the structural level and how the formation of nonphysiological complexes is avoided. Here, we describe structural elements called interface add-ons that fulfill this function and elucidate their role for the diversification of protein–protein interactions during evolution. We identified interface add-ons in 10% of a representative set of bacterial, heteromeric protein complexes. The importance of interface add-ons for protein–protein interaction specificity is demonstrated by an exemplary experimental characterization of over 30 cognate and hybrid glutamine amidotransferase complexes in combination with comprehensive genetic profiling and protein design. Moreover, growth experiments showed that the lack of interface add-ons can lead to physiologically harmful cross-talk between essential biosynthetic pathways. In sum, our complementary in silico, in vitro, and in vivo analysis argues that interface add-ons are a practical and widespread evolutionary strategy to prevent the formation of nonphysiological complexes by specializing protein–protein interactions.

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals The disordered P granule protein LAF-1 drives phase separation into droplets with tunable viscosity and dynamics

2015 ◽  
Vol 112 (23) ◽  
pp. 7189-7194 ◽  
Author(s):  
Shana Elbaum-Garfinkle ◽  
Younghoon Kim ◽  
Krzysztof Szczepaniak ◽  
Carlos Chih-Hsiung Chen ◽  
Christian R. Eckmann ◽  
...  
Keyword(s):  
Phase Separation ◽  
Phase Boundary ◽  
Single Molecule ◽  
Protein Interactions ◽  
Driving Forces ◽  
Early Embryo ◽  
P Granules ◽  
Intrinsically Disordered

P granules and other RNA/protein bodies are membrane-less organelles that may assemble by intracellular phase separation, similar to the condensation of water vapor into droplets. However, the molecular driving forces and the nature of the condensed phases remain poorly understood. Here, we show that the Caenorhabditis elegans protein LAF-1, a DDX3 RNA helicase found in P granules, phase separates into P granule-like droplets in vitro. We adapt a microrheology technique to precisely measure the viscoelasticity of micrometer-sized LAF-1 droplets, revealing purely viscous properties highly tunable by salt and RNA concentration. RNA decreases viscosity and increases molecular dynamics within the droplet. Single molecule FRET assays suggest that this RNA fluidization results from highly dynamic RNA–protein interactions that emerge close to the droplet phase boundary. We demonstrate than an N-terminal, arginine/glycine rich, intrinsically disordered protein (IDP) domain of LAF-1 is necessary and sufficient for both phase separation and RNA–protein interactions. In vivo, RNAi knockdown of LAF-1 results in the dissolution of P granules in the early embryo, with an apparent submicromolar phase boundary comparable to that measured in vitro. Together, these findings demonstrate that LAF-1 is important for promoting P granule assembly and provide insight into the mechanism by which IDP-driven molecular interactions give rise to liquid phase organelles with tunable properties.

scholarly journals A cell-free approach to accelerate the study of protein–protein interactions in vitro

2013 ◽  
Vol 3 (5) ◽  
pp. 20130018 ◽  
Author(s):  
E. Sierecki ◽  
N. Giles ◽  
M. Polinkovsky ◽  
M. Moustaqil ◽  
K. Alexandrov ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Single Molecule ◽  
Protein Interactions ◽  
Protein Complexes ◽  
Protein Interaction Networks ◽  
The Novel ◽  
Protein Protein Interactions ◽  
Single Molecule Fluorescence Spectroscopy ◽  
A Cell

Protein–protein interactions are highly desirable targets in drug discovery, yet only a fraction of drugs act as binding inhibitors. Here, we review the different technologies used to find and validate protein–protein interactions. We then discuss how the novel combination of cell-free protein expression, AlphaScreen and single-molecule fluorescence spectroscopy can be used to rapidly map protein interaction networks, determine the architecture of protein complexes, and find new targets for drug discovery.

scholarly journals Probing DNA interactions with proteins using a single-molecule toolbox: inside the cell, in a test tube and in a computer

2015 ◽  
Vol 43 (2) ◽  
pp. 139-145 ◽  
Author(s):  
Adam J. M. Wollman ◽  
Helen Miller ◽  
Zhaokun Zhou ◽  
Mark C. Leake
Keyword(s):  
Single Molecule ◽  
Protein Interactions ◽  
Super Resolution ◽  
Magnetic Tweezers ◽  
In Vivo Studies ◽  
Live Cells ◽  
Molecular Heterogeneity ◽  
Molecular Signatures

DNA-interacting proteins have roles in multiple processes, many operating as molecular machines which undergo dynamic meta-stable transitions to bring about their biological function. To fully understand this molecular heterogeneity, DNA and the proteins that bind to it must ideally be interrogated at a single molecule level in their native in vivo environments, in a time-resolved manner, fast enough to sample the molecular transitions across the free-energy landscape. Progress has been made over the past decade in utilizing cutting-edge tools of the physical sciences to address challenging biological questions concerning the function and modes of action of several different proteins which bind to DNA. These physiologically relevant assays are technically challenging but can be complemented by powerful and often more tractable in vitro experiments which confer advantages of the chemical environment with enhanced detection signal-to-noise of molecular signatures and transition events. In the present paper, we discuss a range of techniques we have developed to monitor DNA–protein interactions in vivo, in vitro and in silico. These include bespoke single-molecule fluorescence microscopy techniques to elucidate the architecture and dynamics of the bacterial replisome and the structural maintenance of bacterial chromosomes, as well as new computational tools to extract single-molecule molecular signatures from live cells to monitor stoichiometry, spatial localization and mobility in living cells. We also discuss recent developments from our laboratory made in vitro, complementing these in vivo studies, which combine optical and magnetic tweezers to manipulate and image single molecules of DNA, with and without bound protein, in a new super-resolution fluorescence microscope.

scholarly journals Interactions between Papillomavirus L1 and L2 Capsid Proteins

2003 ◽  
Vol 77 (8) ◽  
pp. 4818-4826 ◽  
Author(s):  
Renée L. Finnen ◽  
Kimberly D. Erickson ◽  
Xiaojiang S. Chen ◽  
Robert L. Garcea
Keyword(s):  
Amino Acids ◽  
Capsid Protein ◽  
Single Molecule ◽  
Hydrophobic Interactions ◽  
Protein Complexes ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Hydrophobic Region

ABSTRACT The human papillomavirus (HPV) capsid consists of 360 copies of the major capsid protein, L1, arranged as 72 pentamers on a T=7 icosahedral lattice, with substoichiometric amounts of the minor capsid protein, L2. In order to understand the arrangement of L2 within the HPV virion, we have defined and biochemically characterized a domain of L2 that interacts with L1 pentamers. We utilized an in vivo binding assay involving the coexpression of recombinant HPV type 11 (HPV11) L1 and HPV11 glutathione S-transferase (GST) L2 fusion proteins in Escherichia coli. In this system, L1 forms pentamers, GST=L2 associates with these pentamers, and L1+L2 complexes are subsequently isolated by using the GST tag on L2. The stoichiometry of L1:L2 in purified L1+L2 complexes was 5:1, indicating that a single molecule of L2 interacts with an L1 pentamer. Coexpression of HPV11 L1 with deletion mutants of HPV11 L2 defined an L1-binding domain contained within amino acids 396 to 439 near the carboxy terminus of L2. L2 proteins from eight different human and animal papillomavirus serotypes were tested for their ability to interact with HPV11 L1. This analysis targeted a hydrophobic region within the L1-binding domain of L2 as critical for L1 binding. Introduction of negative charges into this hydrophobic region by site-directed mutagenesis disrupted L1 binding. L1-L2 interactions were not significantly disrupted by treatment with high salt concentrations (2 M NaCl), weak detergents, and urea concentrations of up to 2 M, further indicating that L1 binding by this domain is mediated by strong hydrophobic interactions. L1+L2 protein complexes were able to form virus-like particles in vitro at pH 5.2 and also at pH 6.8, a pH that is nonpermissive for assembly of L1 protein alone. Thus, L1/L2 interactions are primarily hydrophobic, encompass a relatively short stretch of amino acids, and have significant effects upon in vitro assembly.

scholarly journals Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo.

1989 ◽  
Vol 9 (11) ◽  
pp. 4994-5002 ◽  
Author(s):  
M C Blake ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Gene Promoter ◽  
Dhfr Gene ◽  
Recognition Sequence ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Transcription Factor E2f

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.

scholarly journals Transcription factor E2F is required for efficient expression of the hamster dihydrofolate reductase gene in vitro and in vivo

1989 ◽  
Vol 9 (11) ◽  
pp. 4994-5002
Author(s):  
M C Blake ◽  
J C Azizkhan
Keyword(s):  
Dihydrofolate Reductase ◽  
Protein Interactions ◽  
Gene Promoter ◽  
Dhfr Gene ◽  
Recognition Sequence ◽  
Nuclear Extracts ◽  
Efficient Expression ◽  
Transcription Factor E2f

The dihydrofolate reductase (DHFR) gene encodes an enzyme important for metabolism and cell growth. We have found multiple DNA-protein interactions within the hamster DHFR gene promoter in vitro. These interactions occur over the consensus binding sites for two eucaryotic transcription factors. Sp1 and E2F. The DHFR E2F consensus site possesses a dyad symmetry and is unique in its location immediately 3' to the major transcription start site. The interaction of E2F with the DHFR promoter has been detected in HeLa nuclear extracts, confirmed by using partially purified E2F, and characterized by both enzymatic and chemical assays of the DNA-protein interaction. A mutation of the E2F recognition sequence which abolishes E2F binding to the DHFR promoter results in a two- to fivefold decrease of in vitro transcriptional activity and a fivefold reduction of DHFR promoter activity in transient-expression assays. Thus, the interaction of E2F with the DHFR promoter is required for efficient expression of the DHFR gene.

scholarly journals Homeostatic Plasticity Requires Remodeling of the Homer-Shank Interactome

2020 ◽  
Author(s):  
Whitney E. Heavner ◽  
Haley Speed ◽  
Jonathan D. Lautz ◽  
Edward P. Gniffke ◽  
Karen B. Immendorf ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Cortical Neurons ◽  
Barrel Cortex ◽  
Protein Complexes ◽  
Sensory Deprivation ◽  
Interaction Network ◽  
Autism Spectrum ◽  
Homeostatic Plasticity

AbstractNeurons maintain constant levels of excitability using homeostatic scaling, which adjusts relative synaptic strength in response to large changes in overall activity. It is still unknown how homeostatic scaling affects network-level protein interactions in the synapse despite extensive reporting of individual scaling-associated transcriptomic and proteomic changes. Here, we assessed a glutamatergic synapse protein interaction network (PIN) composed of 380 binary interactions among 21 protein members to identify protein complexes altered by synaptic scaling in vitro and in vivo. In cultured cortical neurons, we observed widespread bidirectional PIN alterations during up- and downscaling that reflected rapid glutamate receptor shuttling via synaptic scaffold remodeling. Sensory deprivation of the barrel cortex caused a PIN response that reflected changes in mGluR tone and NMDAR-dependent metaplasticity, consistent with emerging models of homeostatic plasticity in the barrel cortex that restore excitatory/inhibitory balance. Mice lacking Homer1 or Shank3B did not undergo normal PIN rearrangements, suggesting that these Autism Spectrum Disorder (ASD)-linked proteins serve as structural hubs for synaptic homeostasis. Our approach demonstrates how changes in the protein content of synapses during homeostatic plasticity translate into functional PIN alterations that mediate changes in neuron excitability.

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