Memory effects and static disorder reduce information in single-molecule signals

A key theoretical challenge posed by single-molecule studies is the inverse problem of deducing the underlying molecular dynamics from the time evolution of low-dimensional experimental observables. Toward this goal, a variety of low-dimensional models have been proposed as descriptions of single-molecule signals, including random walks with or without conformational memory and/or with static or dynamics disorder. Differentiating among different models presents a challenge, as many distinct physical scenarios lead to similar experimentally observable behaviors such as anomalous diffusion and nonexponential relaxation. Here we show that information-theory-based analysis of single-molecule time series, inspired by Shannon's work studying the information content of printed English, can differentiate between Markov (memoryless) and non-Markov single-molecule signals and between static and dynamic disorder. In particular, non-Markov time series are more predictable and thus can be compressed and transmitted within shorter messages (i.e. have a lower entropy rate) than appropriately constructed Markov approximations, and we demonstrate that in practice the LZMA compression algorithm reliably differentiates between these entropy rates across several simulated dynamical models.

Functionalised Nanopores: Chemical and Biological Modifications

Nanopore technology has established itself as a powerful tool for single-molecule studies. By analysing changes in the ion current flowing through a single transmembrane channel, a wealth of molecular information...

Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

Single-molecule biophysics experiments in silico: Towards a physical model of a replisome

The interpretation of single-molecule experiments is frequently aided by computational modeling of biomolecular dynamics. The growth of computing power and ongoing validation of computational models suggest that it soon may be possible to replace some experiments out-right with computational mimics. Here we offer a blueprint for performing single-molecule studies in silico using a DNA binding protein as a test bed. We demonstrate how atomistic simulations, typically limited to sub-millisecond durations and zeptoliter volumes, can guide development of a coarse-grained model for use in simulations that mimic experimental assays. We show that, after initially correcting excess attraction between the DNA and protein, qualitative consistency between several experiments and their computational equivalents is achieved, while additionally providing a detailed portrait of the underlying mechanics. Finally the model is used to simulate the trombone loop of a replication fork, a large complex of proteins and DNA.

2-photon-fabricated nano-fluidic traps for extended detection of single macromolecules and colloids in solution

The analysis of nanoscopic species, such as proteins and colloidal assemblies, at the single-molecule level has become vital in many areas of fundamental and applied research. Approaches to increase the detection timescales for single molecules in solution without immobilising them onto a substrate surface and applying external fields are much sought after. Here we present an easy-to-implement and versatile nanofluidics-based approach that enables increased observational-timescale analysis of single biomacromolecules and nanoscale colloids in solution. We use two-photon-based hybrid lithography in conjunction with soft lithography to fabricate nanofluidic devices with nano-trapping geometries down to 100 nm in height. We provide a rigorous description and characterisation of the fabrication route that enables the writing of nanoscopic 3D structures directly in photoresist and allows for the integration of nano-trapping and nano-channel geometries within micro-channel devices. Using confocal fluorescence burst detection, we validated the functionality of particle confinement in our nano-trap geometries through measurement of particle residence times. All species under study, including nanoscale colloids, α-synuclein oligomers, and double-stranded DNA, showed a three to five-fold increase in average residence time in the detection volume of nano-traps, due to the additional local steric confinement, in comparison to free space diffusion in a nearby micro-channel. Our approach thus opens-up the possibility for single-molecule studies at prolonged observational timescales to analyse and detect nanoparticles and protein assemblies in solution without the need for surface immobilisation.

Mechanistic Insights From Single-Molecule Studies of Repair of Double Strand Breaks

DNA double strand breaks (DSBs) are among some of the most deleterious forms of DNA damage. Left unrepaired, they are detrimental to genome stability, leading to high risk of cancer. Two major mechanisms are responsible for the repair of DSBs, homologous recombination (HR) and nonhomologous end joining (NHEJ). The complex nature of both pathways, involving a myriad of protein factors functioning in a highly coordinated manner at distinct stages of repair, lend themselves to detailed mechanistic studies using the latest single-molecule techniques. In avoiding ensemble averaging effects inherent to traditional biochemical or genetic methods, single-molecule studies have painted an increasingly detailed picture for every step of the DSB repair processes.