Biology subjects

Brockwell, D. J.

Publications and source records attributed to Brockwell, D. J..

4 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↗

bioRxiv2020

De novo design of transmembrane beta-barrels

The ability of naturally occurring transmembrane {beta}-barrel proteins (TMBs) to spontaneously insert into lipid bilayers and form stable transmembrane pores is a remarkable feat of protein evolution and has been exploited in biotechnology for applications ranging from single molecule DNA and protein sequencing to biomimetic filtration membranes. Because it has not been possible to design TMBs from first principles, these efforts have relied on re-engineering of naturally occurring TMBs that generally have a biological function very different from that desired. Here we leverage the power of de novo computational design coupled with a "hypothesis, design and test" approach to determine principles underlying TMB structure and folding, and find that, unlike almost all other classes of protein, locally destabilizing sequences in both the {beta}-turns and {beta}-strands facilitate TMB expression and global folding by modulating the kinetics of folding and the competition between soluble misfolding and proper folding into the lipid bilayer. We use these principles to design new eight stranded TMBs with sequences unrelated to any known TMB and show that they insert and fold into detergent micelles and synthetic lipid membranes. The designed proteins fold more rapidly and reversibly in lipid membranes than the TMB domain of the model native protein OmpA, and high resolution NMR and X-ray crystal structures of one of the designs are very close to the computational model. The ability to design TMBs from first principles opens the door to custom design of TMBs for biotechnology and demonstrates the value of de novo design to investigate basic protein folding problems that are otherwise hidden by evolutionary history. One sentence summarySuccess in de novo design of transmembrane {beta}-barrels reveals geometric and sequence constraints on the fold and paves the way to design of custom pores for sequencing and other single-molecule analytical applications.

synthetic biology↗

bioRxiv2020

PyXlinkViewer: a flexible tool for visualisation of protein chemical crosslinking data within the PyMOL molecular graphics system

Chemical crosslinking-mass spectrometry (XL-MS) is a valuable technique for gaining insights into protein structure and the organization of macromolecular complexes. XL-MS data yields inter-residue restraints that can be compared with high-resolution structural data. Distances greater than the crosslinker spacer-arm can reveal lowly-populated "excited" states of proteins/protein assemblies, or crosslinks can be used as restraints to generate structural models in the absence of structural data. Despite increasing uptake of XL-MS, there are few tools to enable rapid and facile mapping of XL-MS data onto high-resolution structures or structural models. PyXlinkViewer is a user-friendly plugin for PyMOL v2 that maps intra-protein, inter-protein and dead-end crosslinks onto protein structures/models and automates the calculation of inter-residue distances for the detected crosslinks. This enables rapid visualisation of XL-MS data, assessment of whether a set of detected crosslinks is congruent with structural data, and easy production of high-quality images for publication.

biochemistry↗

bioRxiv2019

Inter-domain dynamics in the chaperone SurA and multi-site binding to its unfolded outer membrane protein clients

The periplasmic chaperone SurA plays a key role in outer membrane protein (OMP) biogenesis. E. coli SurA comprises a core domain and two peptidylprolyl isomerase domains (P1 and P2), but how it binds its OMP clients and the mechanism(s) of its chaperone action remain unclear. Here, we have used chemical cross-linking, hydrogen-deuterium exchange, single-molecule FRET and molecular dynamics simulations to map the client binding site(s) on SurA and to interrogate the role of conformational dynamics of the chaperones domains in OMP recognition. We demonstrate that SurA samples a broad array of conformations in solution in which P2 primarily lies closer to the core/P1 domains than suggested by its crystal structure. Multiple binding sites for OMPs are located primarily in the core domain, with binding of the unfolded OMP resulting in conformational changes between the core/P1 domains. Together, the results portray a model in which unfolded OMP substrates bind in a cradle formed between the SurA domains, with structural flexibility between its domains assisting OMP recognition, binding and release.

biochemistry↗