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

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.

Automated summary

Recent developments in large protein nanopores have allowed for the direct observation and distinction of different conformers of calmodulin, a Ca2+-binding protein. This study demonstrated how a disease-related mutant and different ions can affect the binding capacity and stability of calmodulin. The systematic single-molecule investigation showcased the high sensing resolution offered by the MspA nanopore trap.