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

Abstract

Recent developments concerning large protein nanopores suggest an ew approach to structure profiling of native folded proteins.I nt his work, the large vestibule of Mycobacterium smegmatis porin A( MspA) and calmodulin (CaM), aC a 2+ -binding protein, were used in the direct observation of the protein structure.Three conformers,including the Ca 2+ -free,Ca 2+ -bound, and target peptide-bound states of CaM, were unambiguously distinguished. Adisease related mutant, CaM D129G was also discriminated by MspA, revealing how as ingle amino acid replacement can interfere with the Ca 2+ -binding capacity of the whole protein. The binding capacity and aggregation effect of CaM induced by different ions (Mg 2+ /Sr 2+ /Ba 2+ /Ca 2+ /Pb 2+ /Tb 3+ )w ere 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.

Single‐Molecule Measurement of Carbonic Anhydrase in Cation Coordinated Environment Using MspA Nanopore†

“…[ 18‐19 ] This work provides a new path to de novo design of protein channels with customized transmembrane traffic functions. Besides, relying on the ionic transport, nanopore electro measurement not only exhibits excellent performance for protein profiling, [ 20‐22 ] but also offers opportunities to illustrate phenomena of protein conformational changes, [ 23 ] protein‐protein interaction, [ 24‐25 ] and explore enzyme dynamics in real‐time manner. [ 26‐27 ] Interestingly, nanopore is also possible at this stage, to directly identify the detailed sequence changes taking place during a biosynthesis of a protein, by enzymatic modifications (post‐translational modifications), [ 28‐29 ] and decoding (sequencing).…”