Biology subjects

Wallace, M. I.

Publications and source records attributed to Wallace, M. I..

4 recordsLinked to original sources

bioRxiv2025

Orchestrating Self-Replication in Artificial Cells through Digital Microfluidics

A defining feature of living cells is their ability to self-replicate; but creating artificial cells with this capability remains challenging, due to the complexity of biological division machinery. Rather than seeking to reconstitute this machinery, here we take direct control of DNA replication and compartment division using digital microfluidics. This approach allows us to precisely orchestrate these two fundamental processes, providing insight into how they must be coupled for successful self-replication. Our system achieves autonomous cycles of replication and division, with daughter compartments inheriting parental DNA and maintaining genetic continuity across multiple generations - a key feature of living systems that has been di!cult to achieve in artificial cells. By implementing these processes through direct physical manipulation rather than biochemical complexity, we provide a simple testbed that will help to disentangle the essential requirements for self-replicating systems.

synthetic biology↗

bioRxiv2024

De novo design of α-helical peptide channels with designer stoichiometry

Despite advances in peptide and protein design, the rational design of membrane-spanning peptides that form conducting channels remains challenging due to our imperfect understanding of the sequence-to-structure relationships that drive membrane insertion, assembly, and conductance. Here, we describe the design and computational and experimental characterization of a series of coiled coil-based peptides that form transmembrane -helical barrels. Through a combination of rational and computational design, we obtain barrels with 5 to 7 helices, as characterized in detergent micelles. In lipid bilayers, these peptide assemblies exhibit two conductance states with relative populations dependent on the applied potential: (i) a low-conductance states that correlate with variations in the modeled coiled-coil barrel geometries, indicating stable transmembrane -helical barrels; and (ii) high-conductance states in which single pores change size in discrete steps. Notably, the high-conductance states are similar for all peptides in contrast to the low-conductance states. This indicates the formation of large, dynamic pores through the recruitment and expulsion of peptides, as observed in natural barrel-stave peptide pores. These findings establish rational routes to design and tune functional membrane-spanning peptide channels with specific conductance and geometry.

synthetic biology↗

bioRxiv2021

Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of {beta}-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

biophysics↗

bioRxiv2021

Single-molecule imaging of cholesterol-dependent cytolysin assembly

We exploit single-molecule tracking and optical single channel recording in droplet interface bilayers to resolve the assembly pathway and pore-formation of the archetypical cholesterol-dependent cytolysin nanopore, Perfringolysin O. We follow the stoichiometry and diffusion of Perfringolysin O complexes during assembly with 60 millisecond temporal resolution and 20 nanometre spatial precision. Our results suggest individual nascent complexes can insert into the lipid membrane where they continue active assembly. Overall, these data support a model of stepwise irreversible assembly dominated by monomer addition, but with infrequent assembly from larger partial complexes.

biophysics↗