Allosteric Switching of Calmodulin in a Mycobacterium smegmatis porin A (MspA) Nanopore‐Trap

Abstract

Recent developments concerning large protein nanopores suggest a new approach to structure profiling of native folded proteins. In this work, the large vestibule of Mycobacterium smegmatis porin A (MspA) and calmodulin (CaM), a Ca2+‐binding protein, were used in the direct observation of the protein structure. Three conformers, including the Ca2+‐free, Ca2+‐bound, and target peptide‐bound states of CaM, were unambiguously distinguished. A disease related mutant, CaM D129G was also discriminated by MspA, revealing how a single amino acid replacement can interfere with the Ca2+‐binding capacity of the whole protein. The binding capacity and aggregation effect of CaM induced by different ions (Mg2+/Sr2+/Ba2+/Ca2+/Pb2+/Tb3+) were also investigated and the stability of MspA in extreme conditions was evaluated. This work demonstrates the most systematic single‐molecule investigation of different allosteric conformers of CaM, acknowledging the high sensing resolution offered by the MspA nanopore trap.

A Nanopore‐Based Saccharide Sensor

“…By generating large event amplitudes and rich event features during saccharide sensing, MspA demonstrates a superior performance of saccharide identification in single molecule [20, 25] . According to experimental [29a, 36c, 37] and theoretical assessments, [38] the conical lumen geometry of MspA contributes most to this superior resolution. Discrimination between saccharide isomers or epimers was also demonstrated, further confirming that MspA is structurally superior in saccharide identification.…”

Nanopore Identification of Alditol Epimers and Their Application in Rapid Analysis of Alditol-Containing Drinks and Healthcare Products

“…Nanopore, an emerging single-molecule sensor which provides rapid and sensitive profiling of nucleotides, , amino acids, − biothiols, , neurotransmitter, − nucleic acids, , peptides, , and proteins, − has a great potential to achieve this task. By introducing chemical reactivity into the nanopore lumen, its sensitivity and selectivity could be further improved, disclosing information that is not easily accessed by other means. ,, Phenylboronic acid (PBA), which is known to form covalent bonds with 1,2 or 1,3-diols in aqueous solution, can bind with polyols including sugars − and sugar alcohols. , Recent reports have shown that PBA can serve as a chemically specific adapter of a heterogeneous α-hemolysin (α-HL) permitting the detection of saccharides.…”

Identification of Single-Molecule Catecholamine Enantiomers Using a Programmable Nanopore

“…Biological nanopores are a group of transmembrane porin-based sensors originally developed for sequencing of single molecules. Their high sensing resolution also enables direct probing of metal ions, small molecules, nucleic acids, peptides, and proteins at the level of a single molecule. With their chiral lumen environment and a sufficient resolution of sensing, biological nanopores can, in principle, discriminate between enantiomers when the target molecule is transiently trapped near the pore constriction.…”