Allosteric Switching of Calmodulin in a <i>Mycobacterium smegmatis</i> 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.