Biology subjects

Whitten, A. E.

Publications and source records attributed to Whitten, A. E..

5 recordsLinked to original sources

bioRxiv2025

Tumbleweed: an artificial motor protein that walks along a DNA track

Summary ParagraphMolecular motors are fundamental to life1-6 because of their ability to convert chemical energy into mechanical work, an ability that is conferred by the chemical and structural complexity of their constituent proteins. Scientists have long sought to create artificial protein motors that may reveal insights into how biological motors function. While artificial molecular motors based on small molecules7 and DNA8,9 have been developed, creating an artificial motor protein has remained an elusive goal in synthetic biology10. Here we demonstrate the realization of an artificial protein motor called Tumbleweed (TW) that walks directionally along a DNA track under external control. TW consists of three legs, each with a ligand-gated DNA-binding domain that enables selective interaction with specific sites along a DNA track11. Using single-molecule fluorescence assays and a programmable microfluidic device, we show that TW steps directionally along a designed DNA track in response to a defined sequence of ligand inputs. We built our TW molecular walker using a modular approach, combining existing proteins with known properties to achieve emergent motor function, similar to how Nature evolves new proteins. Our design strategy thus offers a a platform for engineering advanced and dynamic protein functionality. Our demonstration of TW walking represents a step toward developing fully autonomous protein motors and opens new avenues for uncovering and leveraging the principles by which biological motors transduce chemical energy into motion.

biophysics↗

bioRxiv2021

Fine tuning the mechanism of Antigen 43 self-association to modulate aggregation levels of Escherichia coli pathogens

Bacterial aggregates and biofilms allow bacteria to colonise a diverse array of surfaces that can ultimately lead to infections, where the protection they afford permits bacteria to resist anti-microbials and host immune factors. Despite these advantages there is a trade-off, whereby bacterial spread is reduced. As such, biofilm development needs to be regulated appropriately to suit the required niche. Here we investigate members from one of largest groups of bacterial adhesins, the autotransporters, for their critical role in the formation of bacterial aggregates and biofilms. We describe the structural and functional characterisation of autotransporter Ag43 homologues from diverse pathogenic Escherichia strains. We reveal a common mode of trans-association that leads to cell clumping and show that subtle variations in these interactions governs their aggregation kinetics. Our in depth investigation reveals an underlying molecular basis for the tuning of bacterial aggregation.

biochemistry↗

bioRxiv2018

Engineered variants provide new insight into the structural properties important for activity of the highly dynamic, trimeric protein disulfide isomerase, PmScsC

Suppressor of copper sensitivity protein C from Proteus mirabilis (PmScsC) is a homotrimeric disulfide isomerase that plays a role in copper tolerance - a key virulence trait of the uropathogen. Each protomer of the enzyme has an N-terminal trimerisation stem (59 residues) containing a flexible linker (11 residues) connected to a thioredoxin-fold-containing catalytic domain (163 residues). Here, we characterise two PmScsC variants, PmScsC{Delta}N and PmScsC{Delta}Linker. PmScsC{Delta}N, is an N-terminally truncated form of the protomer with two helices of the trimerisation stem removed, generating a protein with dithiol oxidase rather than disulfide isomerase activity. The crystal structure of PmScsC{Delta}N reported here reveals - as expected - a monomer that is structurally similar to the catalytic domain of native PmScsC. The second variant PmScsC{Delta}Linker was designed to remove the 11 amino acid linker and we show that it generates a protein that has neither disulfide isomerase nor dithiol oxidase activity. The crystal structure of PmScsC{Delta}Linker reveals a trimeric arrangement, with the catalytic domains packed together very closely. Small angle X-ray scattering analysis found that native PmScsC is predominantly trimeric in solution even at low concentration, whereas PmScsC{Delta}Linker exists as an equilibrium between monomeric, dimeric and trimeric states, with the monomeric form dominating at low concentrations. These findings increase our understanding of disulfide isomerase activity, showing how (i) oligomerisation, (ii) spacing between, and (iii) dynamic motion of, catalytic domains in PmScsC all contribute to its native function.

biochemistry↗

bioRxiv2018

The atypical thiol-disulfide exchange protein α-DsbA2 from Wolbachia pipientis is a homotrimeric disulfide isomerase

DiSulfide Bond (DSB) oxidative folding enzymes are master regulators of virulence localized to the periplasm of many Gram-negative bacteria. The archetypal DSB machinery from Escherichia coli K12 has a dithiol oxidizing redox relay pair (DsbA/B), a disulfide isomerizing redox relay pair (DsbC/D) and specialist reducing enzymes DsbE and DsbG that also interact with DsbD. By contrast the Gram-negative bacterium Wolbachia pipientis encodes just three DSB enzymes. Two of these -DsbA1 and -DsbB form a redox relay pair analogous to E. coli DsbA/B. The third enzyme -DsbA2 incorporates a DsbA-like sequence but does not interact with -DsbB. In comparison with other DsbA enzymes, -DsbA2 has [~]50 extra N-terminal residues. The crystal structure of -DsbA2{Delta}N, the N-terminally truncated form in which these residues are removed confirms the DsbA-like nature of this domain. However, -DsbA2 does not have DsbA-like activity: it is structurally and functionally different as a consequence of its N-terminal residues. First, -DsbA2 is a powerful disulfide isomerase and a poor dithiol oxidase - ie its role is to shuffle rather than introduce disulfide bonds. Moreover, small-angle X-ray scattering of -DsbA2 reveals a homotrimeric arrangement. Our results allow us to draw conclusions about the factors required for functionally equivalent enzymatic activity across structurally diverse protein architectures.

biochemistry↗

bioRxiv2018

Identification of a novel tetrameric structure for human apolipoprotein-D

Apolipoprotein-D is a 25 kDa glycosylated member of the lipocalin family that folds into an eight-stranded {beta}-barrel with a single adjacent -helix. Apolipoprotein-D specifically binds a range of small hydrophobic ligands such as progesterone and arachidonic acid and has an antioxidant function that is in part due to the reduction of peroxidised lipids by methionine-93. Therefore, apolipoprotein-D plays multiple roles throughout the body and is protective in Alzheimers disease, where apolipoprotein-D overexpression reduces the amyloid-{beta} burden in Alzheimers disease mouse models.\n\nOligomerisation is a common feature of lipocalins that can influence ligand binding. The native structure of apolipoprotein-D, however, has not been conclusively defined. Apolipoprotein-D is generally described as a monomeric protein, although it dimerises when reducing peroxidised lipids.\n\nHere, we investigated the native structure of apolipoprotein-D derived from plasma, breast cyst fluid (BCF) and cerebrospinal fluid. In plasma and cerebrospinal fluid, apolipoprotein-D was present in high-molecular weight complexes, potentially in association with lipoproteins. In contrast, apolipoprotein-D in BCF formed distinct oligomeric species. We assessed apolipoprotein-D oligomerisation using native apolipoprotein-D purified from BCF and a suite of complementary methods, including multi-angle laser light scattering, analytical ultracentrifugation and small-angle X-ray scattering. Our analyses showed that apolipoprotein-D predominantly forms a {bsim}95 to {bsim}100 kDa tetramer. Small-angle X-ray scattering analysis confirmed these findings and provided a structural model for apolipoprotein-D tetramer. These data indicate apolipoprotein-D rarely exists as a free monomer under physiological conditions and provide insights into novel native structures of apolipoprotein-D and into oligomerisation behaviour in the lipocalin family.

biochemistry↗