49 research outputs found
Self-Assembling Protein Surfaces for In Situ Capture of Cell-Free-Synthesized Proteins
We present a new method for the surface capture of proteins in cell-free protein synthesis (CFPS). We demonstrate the spontaneous self-assembly of the protein BslA into functionalizable surfaces on the surface of a CFPS reaction chamber. We show that proteins can be covalently captured by such surfaces, using âCatcher/Tagâ technology. Importantly, proteins of interest can be captured either when synthesised in situ by CFPS above the BslA surfaces, or when added as pure protein. The simplicity and cost efficiency of this method suggest that it will find many applications in cell-free-based methods
Liveâcell superâresolution imaging of actin using LifeActâ14 with a PAINTâbased approach
We present directâLIVEâPAINT, an easyâtoâimplement approach for the nanoscopic imaging of protein structures in live cells using labeled binding peptides. We demonstrate the feasibility of directâLIVEâPAINT with an actinâbinding peptide fused to EGFP, the location of which can be accurately determined as it transiently binds to actin filaments. We show that directâLIVEâPAINT can be used to image actin structures below the diffractionâlimit of light and have used it to observe the dynamic nature of actin in live cells. We envisage a similar approach could be applied to imaging other proteins within live mammalian cells
Single-molecule FRET studies on alpha-synuclein oligomerization of Parkinson's disease genetically related mutants.
Oligomers of alpha-synuclein are toxic to cells and have been proposed to play a key role in the etiopathogenesis of Parkinson's disease. As certain missense mutations in the gene encoding for alpha-synuclein induce early-onset forms of the disease, it has been suggested that these variants might have an inherent tendency to produce high concentrations of oligomers during aggregation, although a direct experimental evidence for this is still missing. We used single-molecule Förster Resonance Energy Transfer to visualize directly the protein self-assembly process by wild-type alpha-synuclein and A53T, A30P and E46K mutants and to compare the structural properties of the ensemble of oligomers generated. We found that the kinetics of oligomer formation correlates with the natural tendency of each variant to acquire beta-sheet structure. Moreover, A53T and A30P showed significant differences in the averaged FRET efficiency of one of the two types of oligomers formed compared to the wild-type oligomers, indicating possible structural variety among the ensemble of species generated. Importantly, we found similar concentrations of oligomers during the lag-phase of the aggregation of wild-type and mutated alpha-synuclein, suggesting that the properties of the ensemble of oligomers generated during self-assembly might be more relevant than their absolute concentration for triggering neurodegeneration.LT has been recipient of a grant PAT Post Doc Outgoing 2009 â 7th Framework Program Marie Curie COFUND actions. NC was funded by a Royal Society Dorothy Hodgkin Research Fellowship and is currently a RamĂłn y Cajal Research Fellow (Spanish Ministry of Economy and Competitiveness). MHH thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. AJD is funded by the Schiff Foundation.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/srep1669
Synaptic expression of TAR-DNA-binding protein 43 in the mouse spinal cord determined using super-resolution microscopy
Funding: This work was supported by Motor Neurone Disease (MND) Association UK (Miles/Apr18/863-791), Chief Scientist Office, RS Macdonald Charitable Trust, ALS CURE Project, the European Research Council (ERC) under the European Unionâs Horizon 2020 Research and Innovation Programme (695568 SYNNOVATE), Simons Foundation Autism Research Initiative (529085), and the Wellcome Trust (Technology Development grant 202932).Amyotrophic Lateral Sclerosis (ALS) is characterised by a loss of motor neurons in the brain and spinal cord that is preceded by early-stage changes in synapses that may be associated with TAR-DNA-Binding Protein 43 (TDP-43) pathology. Cellular inclusions of hyperphosphorylated TDP-43 (pTDP-43) are a key hallmark of neurodegenerative diseases such ALS. However, there has been little characterisation of the synaptic expression of TDP-43 inside subpopulations of spinal cord synapses. This study utilises a range of high-resolution and super-resolution microscopy techniques with immunolabelling, as well as an aptamer-based TDP-43 labelling strategy visualised with single-molecule localisation microscopy, to characterise and quantify the presence of pTDP-43 in populations of excitatory synapses near where motor neurons reside in the lateral ventral horn of the mouse lumbar spinal cord. We observe that TDP-43 is expressed in approximately half of spinal cord synapses as nanoscale clusters. Synaptic TDP-43 clusters are found most abundantly at synapses associated with VGLUT1-positive presynaptic terminals, compared to VGLUT2-associated synapses. Our nanoscopy techniques showed no difference in the subsynaptic expression of pTDP-43 in the ALS mouse model, SOD1G93a, compared to healthy controls, despite prominent structural deficits in VGLUT1-associated synapses in SOD1G93a mice. This research characterizes the basic synaptic expression of TDP-43 with nanoscale precision and provides a framework with which to investigate the potential relationship between TDP-43 pathology and synaptic pathology in neurodegenerative diseases.Publisher PDFPeer reviewe
Determining the location of the α-synuclein dimer Interface using native top-down fragmentation and isotope depletion-mass spectrometry
α-Synuclein
(αSyn), a 140-residue intrinsically disordered
protein, comprises the primary proteinaceous component of pathology-associated
Lewy body inclusions in Parkinsonâs disease (PD). Due to its
association with PD, αSyn is studied extensively; however, the
endogenous structure and physiological roles of this protein are yet
to be fully understood. Here, ion mobility-mass spectrometry and native
top-down electron capture dissociation fragmentation have been used
to elucidate the structural properties associated with a stable, naturally
occurring dimeric species of αSyn. This stable dimer appears
in both wild-type (WT) αSyn and the PD-associated variant A53E.
Furthermore, we integrated a novel method for generating isotopically
depleted protein into our native top-down workflow. Isotope depletion
increases signal-to-noise ratio and reduces the spectral complexity
of fragmentation data, enabling the monoisotopic peak of low abundant
fragment ions to be observed. This enables the accurate and confident
assignment of fragments unique to the αSyn dimer to be assigned
and structural information about this species to be inferred. Using
this approach, we were able to identify fragments unique to the dimer,
which demonstrates a C-terminal to C-terminal interaction between
the monomer subunits. The approach in this study holds promise for
further investigation into the structural properties of endogenous
multimeric species of αSyn
A mechanistic model of tau amyloid aggregation based on direct observation of oligomers.
Protein aggregation plays a key role in neurodegenerative disease, giving rise to small oligomers that may become cytotoxic to cells. The fundamental microscopic reactions taking place during aggregation, and their rate constants, have been difficult to determine due to lack of suitable methods to identify and follow the low concentration of oligomers over time. Here we use single-molecule fluorescence to study the aggregation of the repeat domain of tau (K18), and two mutant forms linked with familial frontotemporal dementia, the deletion mutant ÎK280 and the point mutant P301L. Our kinetic analysis reveals that aggregation proceeds via monomeric assembly into small oligomers, and a subsequent slow structural conversion step before fibril formation. Using this approach, we have been able to quantitatively determine how these mutations alter the aggregation energy landscape.D.K. acknowledges funding from the Wellcome Trust (WT089703) and MRC. E.M. acknowledges funding from the Wellcome Trust (WT089703), DZNE and Max-Planck-Society. M.K. acknowledges fellowships from the Danish research council and the Lundbeck Foundation. N.S. and M.H.H acknowledge funding from the Augustus Newman foundation. G.A.G is funded by the Schiff Foundation. T.P.J.K acknowledges funding from the ERC, Augustus Newman Foundation and the BBSRC.This is the final version of the article. It first appeared from NPG via http://dx.doi.org/10.1038/ncomms802
Alpha-Synuclein Oligomers Interact with Metal Ions to Induce Oxidative Stress and Neuronal Death in Parkinson's Disease
Protein aggregation and oxidative stress are both key pathogenic processes in Parkinson's disease, although the mechanism by which misfolded proteins induce oxidative stress and neuronal death remains unknown. In this study, we describe how aggregation of alpha-synuclein (α-S) from its monomeric form to its soluble oligomeric state results in aberrant free radical production and neuronal toxicity
Fast flow microfluidics and single-molecule fluorescence for the rapid characterization of α-synuclein oligomers.
α-Synuclein oligomers can be toxic to cells and may be responsible for cell death in Parkinson's disease. Their typically low abundance and highly heterogeneous nature, however, make such species challenging to study using traditional biochemical techniques. By combining fast-flow microfluidics with single-molecule fluorescence, we are able to rapidly follow the process by which oligomers of αS are formed and to characterize the species themselves. We have used the technique to show that populations of oligomers with different FRET efficiencies have varying stabilities when diluted into low ionic strength solutions. Interestingly, we have found that oligomers formed early in the aggregation pathway have electrostatic repulsions that are shielded in the high ionic strength buffer and therefore dissociate when diluted into lower ionic strength solutions. This property can be used to isolate different structural groups of αS oligomers and can help to rationalize some aspects of αS amyloid fibril formation.M.H.H. thanks the Royal Society of Chemistry (Analytical Chemistry Trust Fund) for his studentship. L.T. has been the recipient of a grant PAT Post Doc Outgoing 2009 â 7th Framework Program Marie Curie COFUND actions. A.J.D. is funded by the Schiff Foundation.This is the author accepted manuscript. The final version is available from ACS via http://dx.doi.org/10.1021/acs.analchem.5b0181