Biology subjects

Radford, S.

Publications and source records attributed to Radford, S..

5 recordsLinked to original sources

bioRxiv2024

Residues 2-7 of alpha-synuclein regulate amyloid formation via lipid-dependent and -independent pathways

Amyloid formation by -synuclein (Syn) occurs in Parkinsons disease, multiple system atrophy, and dementia with Lewy bodies. Deciphering the residues that regulate Syn amyloid fibril formation will not only provide mechanistic insight, but may also reveal new targets to prevent and treat disease. Previous investigations have identified several regions of Syn to be important in the regulation of amyloid formation, including the non-amyloid-{beta} component (NAC), P1 region (residues 36-42), and residues in the C-terminal domain. Recent studies have also indicated the importance of the N-terminal region of Syn for both its physiological and pathological roles. Here, the role of residues 2-7 in the N-terminal region of Syn are investigated in terms of their ability to regulate amyloid fibril formation in vitro and in vivo. Deletion of these residues (Syn{Delta}N7) slows the rate of fibril formation in vitro and reduces the capacity of the protein to be recruited by wild-type (SynWT) fibril seeds, despite cryo-EM showing a fibril structure consistent with those of full-length Syn. Strikingly, fibril formation of Syn{Delta}N7 is not induced by liposomes, despite the protein binding to liposomes with similar affinity to SynWT. A Caenorhabditis elegans model also showed that Syn{Delta}N7::YFP forms few puncta and lacks motility and lifespan defects typified by expression of SynWT::YFP. Together, the results demonstrate the involvement of residues 2-7 of Syn in amyloid formation, revealing a new target for the design of amyloid inhibitors that may leave the functional role of the protein in membrane binding unperturbed. Significance StatementAmyloid formation of -synuclein (Syn) is associated with Parkinsons disease. Attempts to target Syn aggregation to treat synucleinopathies, thus far, have been unsuccessful. A better understanding of residues that regulate amyloid formation may reveal new targets for therapeutics. Here, six residues at the N-terminus of Syn are identified as regulators of amyloid formation. Deletion of these residues slows lipid-independent assembly, ablates lipid-dependent amyloid formation in vitro, and prevents aggregation and its associated cellular toxicity in vivo. Importantly, these residues are not necessary for binding to synthetic membranes. The work reveals a new target for the prevention of synucleinopathies by disfavouring aggregation without perturbing membrane binding, a property considered to be essential for the physiological function of Syn at the synapse.

biophysics↗

bioRxiv2024

Single molecule delivery into living cells

Controlled manipulation of cultured cells by delivery of exogenous macromolecules is a cornerstone of experimental biology. Here we describe a platform which uses nanopipettes to deliver defined numbers of macromolecules into cultured cell lines and primary cells at single molecule resolution. In the nanoinjection platform the nanopipette is used as both a scanning ion conductance microscope (SICM) scanning probe and as an injection probe. The SICM is used to position the nanopipette above the cell surface, before the nanopipette is inserted into the cell into a defined location and to a predefined depth. We demonstrate that the nanoinjection platform enables the quantitative delivery of DNA, globular proteins and protein fibrils into cells with single molecule resolution and that delivery results in a phenotypic change in the cell that depends on the identity of the molecules introduced. Using experiments and computational modelling, we also show that macromolecular crowding in the cell increases the signal to noise ratio for the detection of translocation events, thus the cell itself enhances the detection of the molecules delivered.

biophysics↗

bioRxiv2023

Sculpting conducting nanopore size and shape through de novo protein design

Transmembrane {beta}-barrels (TMBs) are widely used for single molecule DNA and RNA sequencing and have considerable potential for a broad range of sensing and sequencing applications. Current engineering approaches for nanopore sensors are limited to naturally occurring channels such as CsgG, which have evolved to carry out functions very different from sensing, and hence provide sub-optimal starting points. In contrast, de novo protein design can in principle create an unlimited number of new nanopores with any desired properties. Here we describe a general approach to the design of transmembrane {beta}-barrel pores with different diameter and pore geometry. NMR and crystallographic characterization shows that the designs are stably folded with structures close to the design models. We report the first examples of de novo designed TMBs with 10, 12 and 14 stranded {beta}-barrels. The designs have distinct conductances that correlate with their pore diameter, ranging from 110 pS ([~]0.5 nm pore diameter) to 430 pS ([~]1.1 nm pore diameter), and can be converted into sensitive small-molecule sensors with high signal to noise ratio. The capability to generate on demand {beta}-barrel pores of defined geometry opens up fundamentally new opportunities for custom engineering of sequencing and sensing technologies. One sentence summaryDe novo design enables the generation of stable and quite transmembrane beta-barrel nanopores with tailored sizes, shapes and properties.

synthetic biology↗

bioRxiv2023

The structural architecture of an α-synuclein toxic oligomer

Oligomeric species populated during -synuclein aggregation are considered key drivers of neurodegeneration in Parkinsons disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. PSM3 is a nanomolar peptide binder of -synuclein oligomers that inhibits aggregation by blocking oligomer to fibril conversion. Here, we investigate the binding of PSM3 to -synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSM3 selectively targets an -synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the monomeric and oligomeric states. This -synuclein region plays a pivotal role in oligomer to fibril conversion, as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early-onset Parkinsons disease, causes a conformational fluctuation in this region, leading to delayed oligomer to fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in -synuclein oligomers, advance our comprehension of oligomer to amyloid fibril conversion and reveal a new facet of -synuclein pathogenic mutations.

biophysics↗

bioRxiv2022

Asymmetric Ion Mobility and Interface Displacement Drive the Signal Enhancement in a Polymer-electrolyte Nanopore

Solid-state nanopores have been widely employed in the detection of biomolecules, but low signal-to-noise ratios still represent a major obstacle to enable the discrimination of short nucleic acid and protein sequences. The addition of 50% polyethylene glycol (PEG) to the bath solution was recently demonstrated as a simple way to enhance the detection of such biomolecules translocating through a model solid-state nanopore. Here, we provide a comprehensive description of the physics describing a nanopore measurement carried out in 50% PEG that is supported by finite-element modelling and experiments. We demonstrate that the addition of PEG to the external solution introduces a strong imbalance in the transport properties of cations and anions, drastically affecting the characteristic current response of the nanopore. We further show that the strong asymmetric current response is due to a polarity-dependent ion distribution and transport at the nanopipette tip region, leading to either ion depletion or enrichment for few tens of nanometers across the aperture. Under negative potential, when double-stranded DNA molecules translocate, the depleted region (sensing region) significantly improves the sensitivity compared to systems without PEG. We then introduce a displacement of the interface between pore and external solution to simulate the mechanical interactions between analyte and PEG molecules. We found that this displacement affects the ion distribution in the sensing region, enhancing the detection current during the translocation of biomolecules.

biophysics↗