Biology subjects

Curnow, P.

Publications and source records attributed to Curnow, P..

5 recordsLinked to original sources

bioRxiv2025

Effective sequence-to-expression prediction for membrane proteins using machine learning and computational protein design

The recombinant expression of integral membrane proteins is notoriously challenging. One way to address this challenge is via computational genotype-to-phenotype models that determine how particular sequence features correlate with protein expression levels. However, the potential of such approaches is yet to be fully realised, at least partly because so few expression datasets are available. Here, we study the sequence-to-expression relationships of a library of 12,248 variants of a specific membrane protein derived from combinatorial computational design. The major advantage of this approach lies in the controlled sequence diversity explored in design, making this new dataset directly compatible with lightweight off-the-shelf bioinformatic tools. The expression phenotype of the entire library is assessed in the widely-used recombinant host Escherichia coli. We employed a relatively small dataset of [~]2000 phenotyped sequences to train a sequence-to-expression predictor using supervised machine learning, which achieved high classification accuracy on held-out test sequences. This model was then used to infer the expression of >10,000 unmeasured sequences, and validation of the top predictions of both high and low expressers achieved 100% success rate. Using tools from explainable AI, we identified specific sequence positions and substitutions that are most important in dictating cellular expression levels. This analysis was validated by model-guided protein engineering that achieved an 8-fold increase in the purification yield of a poorly-expressing variant. Our results show that, at least for this controlled dataset, straightforward and interpretable machine learning can reveal the intrinsic sequence code for membrane protein expression.

bioengineering↗

bioRxiv2024

Delineating redox cooperativity in water-soluble and membrane multiheme cytochromes through protein design

Nature has evolved diverse electron transport proteins and multiprotein assemblies essential to the generation and transduction of biological energy. However, substantially modifying or adapting these proteins for user-defined applications or to gain fundamental mechanistic insight can be hindered by their inherent complexity. De novo protein design offers an attractive route to stripping away this confounding complexity, enabling us to probe the fundamental workings of these bioenergetic proteins and systems, while providing robust, modular platforms for constructing completely artificial electron-conducting circuitry. Here, we use a set of de novo designed mono-heme and di-heme soluble and membrane proteins to unpick the contributions of electrostatic micro-environments and dielectric properties of the surrounding protein medium on the inter-heme redox cooperativity that we have previously reported. Experimentally, we find that the two heme sites in both the water-soluble and membrane constructs have broadly equivalent redox potentials in isolation, in agreement with Poisson-Boltzmann Continuum Electrostatics calculations. BioDC, a Python program for the estimation of electron transfer energetics and kinetics within multiheme cytochromes, also predicts equivalent heme sites, and reports that burial within the low dielectric environment of the membrane strengthens heme-heme electrostatic coupling. We conclude that redox cooperativity in our diheme cytochromes is largely driven by heme electrostatic coupling and confirm that this effect is greatly strengthened by burial in the membrane. These results demonstrate that while our de novo proteins present minimalist, new-to-nature constructs, they enable the dissection and microscopic examination of processes fundamental to the function of vital, yet complex, bioenergetic assemblies.

biochemistry↗

bioRxiv2024

Diversity and structure of the deep-sea sponge microbiome in the equatorial Atlantic Ocean

Sponges (phylum Porifera) harbour specific microbial communities that drive the ecology and evolution of the host. Understanding the structure and dynamics of these communities is emerging as a primary focus in marine microbial ecology research. Much of the work to date has focused on sponges from warm and shallow coastal waters, while sponges from the deep ocean remain less well-studied. Here, we present a metataxonomic analysis of the microbial consortia associated with 23 deep-sea sponges. We identify a high abundance of archaea relative to bacteria across these communities, with certain sponge microbiomes comprising more than 90% archaea. Specifically, the archaeal family Nitrosopumilaceae are prolific, comprising over 99% of all archaeal reads. Our analysis revealed sponge microbial communities mirror the host sponge phylogeny, indicating a key role for host taxonomy in defining microbiome composition. Our work confirms the contribution of both evolutionary and environmental processes to the composition of microbial communities in deep-sea sponges. ImportanceThe deep ocean is the largest biome on Earth, accounting for >90% of the planets marine environment. Despite this it remains a largely unexplored ecosystem, with less than 0.01% of the deep seafloor having been quantitatively sampled. Deep-sea sponges are ancient metazoans which harbour complex microbial communities and much still remains to be learned about the composition and diversity of these unique microbiomes. In an effort to address this, here we report a metataxonomic analysis of the microbial consortia associated with 23 deep-sea sponges from the equatorial Atlantic Ocean. Our findings reveal intricate, species-specific microbial communities dominated by ammonia-oxidizing archaea. This study highlights the significant role sponges play in shaping microbial consortia, providing new insights into deep-sea ecosystem dynamics. Importantly, our findings provide a scientific basis for understanding the evolutionary relationships between sponges and their symbiotic microorganisms.

microbiology↗

bioRxiv2022

Cellular production of a de novo membrane cytochrome

Heme-containing integral membrane proteins are at the heart of many bioenergetic complexes and electron transport chains. The importance of these electron relay hubs across biology has inspired the design of de novo proteins that recreate their core features within robust, versatile and tractable protein folds. To this end, we report here the computational design and in-cell production of a minimal diheme membrane cytochrome which successfully integrates into the cellular membrane of live bacteria. This synthetic construct emulates a four-helix bundle found in modern respiratory complexes but has no sequence homology to any polypeptide sequence found in nature. The two b-type hemes, which appear to be recruited from the endogenous heme pool, have distinct split redox potentials with values close to those of natural membrane-spanning cytochromes. The purified protein can engage in rapid biomimetic electron transport with small molecules, with other redox proteins, and with biologically-relevant diffusive electron carriers. We thus report an artificial membrane metalloprotein with the potential to serve as a functional module in electron transfer pathways in both synthetic protocells and living systems.

biochemistry↗

bioRxiv2022

Discovery and biosynthetic assessment of Streptomyces ortus sp nov. isolated from a deep-sea sponge

The deep sea is known to host novel bacteria with the potential to produce a diverse array of undiscovered natural products. Understanding these bacteria is thus of broad interest in ecology and could also underpin applied drug discovery, specifically in the area of antimicrobials. Here, we isolate a new strain of Streptomyces from the tissue of the deep-sea sponge Polymastia corticata collected at a depth of 1869 m from the Gramberg seamount in the Atlantic Ocean. This strain, which was given the initial designation A15ISP2-DRY2T, has a genome size of 9.29 Mb with a GC content of 70.83%. Phylogenomics determined that A15ISP2-DRY2T represents a novel species within the genus Streptomyces as part of the Streptomyces aurantiacus clade. The biosynthetic potential of A15ISP2-DRY2T was assessed relative to other members of the aurantiacus clade via comparative gene cluster family (GCF) analysis. This revealed a clear congruent relationship between phylogeny and GCF content. A15ISP2-DRY2T contains six unique GCFs absent elsewhere in the clade. Culture-based assays were used to demonstrate the antibacterial activity of A15ISP2-DRY2T against two drug-resistant human pathogens. We thus determine A15ISP2-DRY2T to be a novel bacterial species with considerable biosynthetic potential and propose the systematic name Streptomyces ortus sp. nov. Impact StatementThe Streptomyces genus has contributed more to our antibiotic arsenal than any other group of bacteria or fungi. Despite decades of exploration, global analysis has suggested they still possess more undiscovered biosynthetic diversity than any other bacterial group. Isolating novel species of Streptomyces is therefore a priority for antibiotic discovery. Here we isolate a novel strain from a deep-sea sponge and use comparative cluster analysis to identify six biosynthetic clusters unique to our deep-sea strain. This work demonstrates the utility of continuing to isolate novel Streptomyces strains for antibiotic discovery and, for the first time, we used species tree-gene cluster tree reconciliation to assess the contribution of vertical evolution on the biosynthetic gene cluster content of Streptomyces.

genomics↗