Biology subjects

Brinkerhoff, H.

Publications and source records attributed to Brinkerhoff, H..

4 recordsLinked to original sources

bioRxiv2024

Lumen charge governs ion transport in β-barrel nanopores

{beta}-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains in its early stages. In this study, we integrate experimental, numerical, and theoretical approaches to elucidate ion transport mechanisms in these biological nanopores. We identify and characterise two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. Additionally, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for the characterisation and understanding of biological ion transport processes, enabling the design of adaptable biosensors. We illustrate this by optimizing an aerolysin mutant for computing applications, paving the way for novel nanofluidic technologies.

biophysics↗

bioRxiv2024

Resolving sulfation PTMs on a plant peptide hormone using nanopore sequencing

Peptide phytohormones are decorated with post-translational modifications (PTMs) that are crucial for receptor recognition. Tyrosine sulfation on these hormones is essential for plant growth and development1. Measuring the occurrence and position of sulfotyrosine is, however, compromised by major technical challenges during isolation and detection2. We recently introduced a nanopore peptide sequencing method that sensitively detects PTMs at the single-molecule level3. By translocating PTM variants of the plant pentapeptide hormone phytosulfokine (PSK) through a nanopore, we here demonstrate accurate identification of sulfation and phosphorylation on the two tyrosine residues of PSK. Sulfation can be clearly detected and distinguished (>90%) from phosphorylation on the same residue. Moreover, the presence or absence of PTMs on the two close-by tyrosine residues can be accurately determined (>96% accuracy). Our findings demonstrate the extraordinary sensitivity of nanopore protein measurements, providing a new tool for identifying sulfation on peptide phytohormones and promising wider applications to identify protein PTMs.

biophysics↗

bioRxiv2022

Mapping phosphorylation post-translational modifications along single peptides with nanopores

Current methods to detect post-translational modifications (PTMs) of proteins, such as phosphate groups, cannot measure single molecules and often cannot differentiate between closely spaced phosphorylation sites. Using a nanopore sequencing approach, we here report detection of PTMs at the single-molecule level on immunopeptide sequences with cancer-associated phosphate variants. We reliably discriminate peptide sequences with one or two closely spaced phosphates with 95% accuracy for individual reads of single molecules.

biophysics↗

bioRxiv2021

Infinite re-reading of single proteins at single-amino-acid resolution using nanopore sequencing

As identifying proteins is of paramount importance for cell biology and applications, it is of interest to develop a protein sequencer with the ultimate sensitivity of decoding individual proteins. Here, we demonstrate a nanopore-based single-molecule sequencing approach capable of reliably detecting single amino-acid substitutions within individual peptides. A peptide is linked to a DNA molecule that is pulled through the biological nanopore MspA by a DNA helicase in single amino-acid steps. The peptide sequence yields clear stepping ion current signals which allows to discriminate single-amino-acid substitutions in single reads. Molecular dynamics simulations show these signals to result from size exclusion and pore binding. Notably, we demonstrate the capability to rewind peptide reads, obtaining indefinitely many independent reads of the same individual molecule, yielding virtually 100% read accuracy in variant identification, with an error rate less than 10-6. These proof-of-concept experiments constitute a promising basis for developing a single-molecule protein sequencer. One-sentence summaryThis paper presents proof-of-concept experiments and simulations of a nanopore-based approach to sequencing individual proteins.

biophysics↗