Biology subjects

Walker, E. J.

Publications and source records attributed to Walker, E. J..

5 recordsLinked to original sources

bioRxiv2025

Simple high-throughput encoding of deep mutational scanning libraries by oligo-based Golden Gate assembly

Control over mutational library diversity is an essential consideration when engineering proteins, but is often fraught with trade-offs between diversity, specificity, and affordability. Contemporary library assembly approaches often incorporate oligonucleotide pool synthesis to achieve affordable, precise mutagenesis; however, these oligos are often reliant on complex designs to facilitate downstream PCR and/or restriction digests. Direct hybridisation of oligo pools is an overlooked strategy to simplify mutagenesis, especially when paired with a type IIS restriction cloning approach. We validate this approach by designing, hybridising, and deep sequencing single and dual substitution CDR region parts derived from nanobody GA10. Assembly of these parts into a full-length nanobody CDS facilitated the phage display of variant libraries for affinity maturation against its cyclic peptide target. Variants identified through enrichment analysis were expressed in isolation and yielded improved affinities by more than 100-fold. Recent advances in machine learning have successfully inferred improved variants outside of screened library space, but require controlled, multi-mutant libraries. The library assembly approach outlined in this research is well-suited for such approaches.

synthetic biology↗

bioRxiv2025

The Impact of Habitat Impermanence on Metapopulation Viability and Size

Habitat is often impermanent causing the amount and spatial distribution of habitat patches available to species to vary through time. Theory calls for metrics that fully account for the impact of habitat impermanence on metapopulations, yet all use averages that homogenize spatiotemporal impacts of habitat impermanence in some manner. We develop a novel modelling approach using a deterministic variation of the widely used spatially realistic Levins model paired with a continuous time Markov chain to capture the stochastic impacts of habitat impermanence on finite metapopulations. From this model we derive analytic expectations of metapopulation viability and size by weighting landscape capacities and equilibrium occupancies by the quasi-equilibrium distribution of habitat configurations exhibited by landscapes characterized by a simplest form of habitat impermanence (i.e. random and independent habitat patch loss and gain at constant rates). These provide expectations of metapopulation viability and size in absence of transient metapopulation dynamics. We then show how dispersal and colonization/extinction rates, relative to rates of habitat change, separately impact metapopulation viability and size. Using simulations of our model, we show the measures we propose invariably improve estimation of metapopulation viability and size from earlier estimates, but ultimately overestimate both when species less able to keep pace with rates of change from habitat impermanence in the landscapes they occupy. Our research identifies the importance of fully accounting for spatial habitat configurations through time, and highlights the importance of transient dynamics as rates of habitat impermanence increase.

ecology↗

bioRxiv2023

Cooperative conformational transitions and the temperature dependence of enzyme catalysis

Many enzymes display non-Arrhenius behaviour with curved Arrhenius plots in the absence of denaturation. There has been significant debate about the origin of this behaviour and recently the role of the activation heat capacity [Formula] has been widely discussed. If enzyme-catalysed reactions occur with appreciable negative values of [Formula] (arising from narrowing of the conformational space along the reaction coordinate), then curved Arrhenius plots are a consequence. To investigate these phenomena in detail, we have collected high precision temperature-rate data over a wide temperature interval for a model glycosidase enzyme MalL, and a series of mutants that change the temperature-dependence of the enzyme-catalysed rate. We use these data to test a range of models including macromolecular rate theory (MMRT) and an equilibrium model. In addition, we have performed extensive molecular dynamics (MD) simulations to characterise the conformational landscape traversed by MalL in the enzyme-substrate complex and an enzyme-transition state complex. We have crystallised the enzyme in a transition state-like conformation in the absence of a ligand and determined an X-ray crystal structure at very high resolution (1.10 [A]). We show (using simulation) that this enzyme-transition state conformation has a more restricted conformational landscape than the wildtype enzyme. We coin the term "transition state-like conformation (TLC)" to apply to this state of the enzyme. Together, these results imply a cooperative conformational transition between an enzyme-substrate conformation (ES) and a transition-state-like conformation (TLC) that precedes the chemical step. We present a two-state model as an extension of MMRT (MMRT-2S) that describes the data along with a convenient approximation with linear temperature dependence of the activation heat capacity (MMRT-1L) that can be used where fewer data points are available. Our model rationalises disparate behaviour seen for MalL and a thermophilic alcohol dehydrogenase and is consistent with a raft of data for other enzymes. Our model can be used to characterise the conformational changes required for enzyme catalysis and provides insights into the role of cooperative conformational changes in transition state stabilisation that are accompanied by changes in heat capacity for the system along the reaction coordinate. TLCs are likely to be of wide importance in understanding the temperature dependence of enzyme activity, and other aspects of enzyme catalysis.

biochemistry↗

bioRxiv2021

Protein folding stabilities are a major determinant of oxidation rates for buried methionine residues

The oxidation of protein-bound methionines to form methionine sulfoxides has a broad range of biological ramifications, making it important to delineate factors that influence methionine oxidation rates within a protein. This is especially important for biopharmaceuticals, where oxidation can lead to deactivation and degradation. Previously, neighboring residue effects and solvent accessibility (SA) have been shown to impact the susceptibility of methionine residues to oxidation. In this study, we provide proteome-wide evidence that oxidation rates of buried methionine residues are also strongly influenced by the thermodynamic folding stability of proteins. We surveyed the E. coli proteome using several proteomic methodologies and globally measured oxidation rates of methionines in the presence and absence of tertiary structure, as well as folding stabilities of methionine containing domains. The data indicate that buried methionines have a wide range of protection factors against oxidation which correlate strongly with folding stabilities. Concordantly, we show that in comparison to E. coli, the proteome of the thermophile T. thermophilus is significantly more stable and thus more resistant to methionine oxidation. These results indicate that oxidation rates of buried methionines from the native state of proteins can be used as a metric of folding stability. To demonstrate the utility of this correlation, we used native methionine oxidation rates to survey the folding stabilities of E. coli and T. thermophilus proteomes at various temperatures and suggest a model that relates the temperature dependence of the folding stabilities of these two species to their optimal growth temperatures.

biochemistry↗

bioRxiv2018

Global analysis of methionine oxidation provides a census of folding stabilities for the human proteome

The stability of proteins influences their tendency to aggregate, undergo degradation or become modified in cells. Despite their significance to understanding protein folding and function, quantitative analyses of thermodynamic stabilities have been mostly limited to soluble proteins in purified systems. We have used a highly multiplexed proteomics approach, based on analyses of methionine oxidation rates, to quantify stabilities of ~10,000 unique regions within ~3,000 proteins in human cell extracts. The data identify lysosomal and extracellular proteins as the most stable ontological subsets of the proteome. We show that the stability of proteins impacts their tendency to become oxidized and is globally altered by the osmolyte trimethylamine-N-oxide (TMAO). We also show that most proteins designated as intrinsically disordered retain their unfolded structure in the complex environment of the cell. Together, the data provide a census of the stability of the human proteome and validate a methodology for global quantitation of folding thermodynamics.

biochemistry↗