Mutual effects of disorder and order in fusion proteins between intrinsically disordered domains and fluorescent proteins

2012 ◽  
Vol 8 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Marina Lotti ◽  
Sonia Longhi
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Intrinsically Disordered ◽  
Effects Of Disorder

Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain

2018 ◽  
Author(s):  
Sarah Klass ◽  
Matthew J. Smith ◽  
Tahoe Fiala ◽  
Jessica Lee ◽  
Anthony Omole ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fusion Proteins ◽  
Organic Molecules ◽  
Intrinsically Disordered Protein ◽  
Protein Domain ◽  
Enzymatic Cleavage ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Self Assembling ◽  
Protein Tag

Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values below 1 µM being observed in some cases. The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications.

scholarly journals Impact of fluorescent protein fusions on the bacterial flagellar motor

2017 ◽  
Author(s):  
M Heo ◽  
AL Nord ◽  
D Chamousset ◽  
E van Rijn ◽  
HJE Beaumont ◽  
...  
Keyword(s):  
Side Effects ◽  
Protein Function ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Protein Complexes ◽  
Switching Frequency ◽  
Flagellar Motor ◽  
Internal Dynamics ◽  
Biologically Relevant ◽  
Bacterial Flagellar Motor

AbstractFluorescent fusion proteins open a direct and unique window onto protein function. However, they also introduce the risk of perturbation of the function of the native protein. Successful applications of fluorescent fusions therefore rely on a careful assessment and minimization of the side effects. Such insight, however, is still lacking for many applications of fluorescent fusions. This is particularly relevant in the study of the internal dynamics of motor protein complexes, where both the chemical and mechanical reaction coordinates can be affected. Fluorescent proteins fused to thestatorof the bacterial flagellar motor (BFM) complex have previously been used to successfully unveil the internal subunit dynamics of the motor. Here we report the effects of three different fluorescent proteins fused to the stator, all of which altered BFM behavior. The torque generated by individual stators was reduced while their stoichiometry in the complex remained unaffected. MotB fusions decreased the rotation-direction switching frequency of single motors and induced a novel BFM behavior: a bias-dependent asymmetry in the speed attained in the two rotation directions. All these effects could be mitigated by the insertion of a linker at the fusion point. These findings provide a quantitative account of the effects of fluorescent fusions on BFM dynamics and their alleviation—new insights that advance the use of fluorescent fusions to probe the dynamics of protein complexes.Author summaryMuch of what is known about the biology of proteins was discovered by fusing them to fluorescent proteins that allow detection of their location. But the label comes at a cost: the presence of the tag can alter the behavior of the protein of interest in unforeseen, yet biologically relevant ways. These side effects limit the depth to which fluorescent proteins can be used to probe protein function. One of the systems that has been successfully studied with fluorescent fusions for which these effects have not been addressed are dynamic protein complexes that carry out mechanical work. We examined how fluorescent proteins fused to a component of the bacterial flagellar motor complex impacts its function. Our findings show that the fusion proteins altered biologically relevant dynamical properties of the motor, including induction of a novel mechanical behavior, and demonstrate an approach to alleviate this. These results advance our ability to dissect the bacterial flagellar motor, and the internal dynamics of protein complexes in general, with fluorescent fusion proteins while causing minimal perturbation.

scholarly journals Synthetic and genetic dimers as quantification ruler for single-molecule counting with PALM

2019 ◽  
Vol 30 (12) ◽  
pp. 1369-1376 ◽  
Author(s):  
Tim N. Baldering ◽  
Marina S. Dietz ◽  
Karl Gatterdam ◽  
Christos Karathanasis ◽  
Ralph Wieneke ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Superresolution Microscopy ◽  
Superresolution Imaging ◽  
Molecular Information ◽  
Kinetics Of

How membrane proteins oligomerize determines their function. Superresolution microscopy can report on protein clustering and extract quantitative molecular information. Here, we evaluate the blinking kinetics of four photoactivatable fluorescent proteins for quantitative single-molecule microscopy. We identified mEos3.2 and mMaple3 to be suitable for molecular quantification through blinking histogram analysis. We designed synthetic and genetic dimers of mEos3.2 as well as fusion proteins of monomeric and dimeric membrane proteins as reference structures, and we demonstrate their versatile use for quantitative superresolution imaging in vitro and in situ. We further found that the blinking behavior of mEos3.2 and mMaple3 is modified by a reducing agent, offering the possibility to adjust blinking parameters according to experimental needs.

A simple and effective method for protein subcellular localization using Agrobacterium-mediated transformation of onion epidermal cells

Biologia ◽  
2007 ◽  
Vol 62 (5) ◽  
Author(s):  
Wei Sun ◽  
Ziyi Cao ◽  
Yan Li ◽  
Yanxiu Zhao ◽  
Hui Zhang
Keyword(s):  
Subcellular Localization ◽  
Particle Bombardment ◽  
Fusion Proteins ◽  
Fluorescent Proteins ◽  
Transformed Cells ◽  
Protein Subcellular Localization ◽  
Transformation Protocol ◽  
Agrobacterium Mediated Transformation ◽  
The Mean ◽  
Onion Epidermal Cells

AbstractA modified Agrobacterium-mediated transformation protocol has been successfully used for transient expression of the intrinsically fluorescent proteins and their fusion proteins in onion epidermis. The mean of the transformed cells rate per peel is about 10.5±0.9%, while that of the particle bombardment method is at the range 2.0±0.4%. To compare with the prevailing method of micro-projectile bombardment, the modified Agrobacterium-mediated transformation may provide with higher efficiency and even more simplified manipulability on a lower budget.

Properties of Baculovirus Particles Displaying GFP Analyzed by Fluorescence Correlation Spectroscopy

2002 ◽  
Vol 383 (12) ◽  
pp. 1941-1946 ◽  
Author(s):  
J. Toivola ◽  
K. Ojala ◽  
P.O. Michel ◽  
M. Vuento ◽  
C. Oker-Blom
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Sodium Dodecyl ◽  
Recombinant Baculovirus ◽  
Mean Value ◽  
Correlation Spectroscopy ◽  
Viral Envelope ◽  
Fluorescence Correlation

Abstract Recombinant baculovirus particles displaying green fluorescent protein (GFP) fused to the major envelope glycoprotein gp64 of the Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV) were characterized by fluorescence correlation spectroscopy (FCS). FCS detected Brownian motion of single, intact recombinant baculovirus display particles with a diffusion coefficient (D) of (2.89±0.74)10 8 cm2s 1 and an apparent hydrodynamic radius of 83.35±21.22 nm. In the presence of sodium dodecyl sulfate (SDS), Triton X-100, and octylglucoside, the diffusion time was reduced to the 0.2 ms range (D = 7.5710 7 cm2s 1), showing that the fusion proteins were anchored in the viral envelope. This allowed for a calculation of the number of single gp64 fusion proteins incorporated in the viral membrane. A mean value of 3.2 fluorescent proteins per virus particle was obtained. Our results show that FCS is the method of choice for studying enveloped viruses such as a display virus with one component being GFP.

scholarly journals Development of a single-stranded DNA-binding protein fluorescent fusion toolbox

2020 ◽  
Vol 48 (11) ◽  
pp. 6053-6067
Author(s):  
Katarzyna Dubiel ◽  
Camille Henry ◽  
Lisanne M Spenkelink ◽  
Alexander G Kozlov ◽  
Elizabeth A Wood ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Binding ◽  
Single Molecule ◽  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Single Stranded Dna ◽  
Intrinsically Disordered ◽  
C Terminus ◽  
Sites Of Action

Abstract Bacterial single-stranded DNA-binding proteins (SSBs) bind single-stranded DNA and help to recruit heterologous proteins to their sites of action. SSBs perform these essential functions through a modular structural architecture: the N-terminal domain comprises a DNA binding/tetramerization element whereas the C-terminus forms an intrinsically disordered linker (IDL) capped by a protein-interacting SSB-Ct motif. Here we examine the activities of SSB-IDL fusion proteins in which fluorescent domains are inserted within the IDL of Escherichia coli SSB. The SSB-IDL fusions maintain DNA and protein binding activities in vitro, although cooperative DNA binding is impaired. In contrast, an SSB variant with a fluorescent protein attached directly to the C-terminus that is similar to fusions used in previous studies displayed dysfunctional protein interaction activity. The SSB-IDL fusions are readily visualized in single-molecule DNA replication reactions. Escherichia coli strains in which wildtype SSB is replaced by SSB-IDL fusions are viable and display normal growth rates and fitness. The SSB-IDL fusions form detectible SSB foci in cells with frequencies mirroring previously examined fluorescent DNA replication fusion proteins. Cells expressing SSB-IDL fusions are sensitized to some DNA damaging agents. The results highlight the utility of SSB-IDL fusions for biochemical and cellular studies of genome maintenance reactions.

Self-Assembling Micelles Based on an Intrinsically Disordered Protein Domain

2018 ◽  
Author(s):  
Sarah Klass ◽  
Matthew J. Smith ◽  
Tahoe Fiala ◽  
Jessica Lee ◽  
Anthony Omole ◽  
...  
Keyword(s):  
Drug Delivery ◽  
Fusion Proteins ◽  
Organic Molecules ◽  
Intrinsically Disordered Protein ◽  
Protein Domain ◽  
Enzymatic Cleavage ◽  
Intrinsically Disordered ◽  
Disordered Protein ◽  
Self Assembling ◽  
Protein Tag

Herein, we describe a new series of fusion proteins that have been developed to self-assemble spontaneously into stable micelles that are 27 nm in diameter after enzymatic cleavage of a solubilizing protein tag. The sequences of the proteins are based on a human intrinsically disordered protein, which has been appended with a hydrophobic segment. The micelles were found to form across a broad range of pH, ionic strength, and temperature conditions, with critical micelle concentration (CMC) values below 1 µM being observed in some cases. The reported micelles were found to solubilize hydrophobic metal complexes and organic molecules, suggesting their potential suitability for catalysis and drug delivery applications.

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