Mapping Potential Engineering Sites of Mycobacterium smegmatis porin A (MspA) to Form a Nanoreactor

Protein nanopores can be engineered as nanoreactors to investigate single-molecule chemical reactions. Recent studies have demonstrated that Mycobacterium smegmatis porin A (MspA) nanopore is a superior engineering template acknowledging its geometrical advantages. However, reported engineering of MspA to form a nanoreactor has focused only on site 91 and mapping of other engineering sites have never been performed before. By taking tetrachloraurate(III) ([AuCl₄]^-) as a model reactant, potential engineering sites within the pore constriction of MspA have been thoroughly investigated. It is discovered that the produced event amplitude is inversely correlated to the cross-sectional diameter of the pore constriction size at the engineering site, providing evidence that site 91 is actually already the optimum place to introduce the chemical reactivity. Other unavailable engineering sites, which either significantly interfere with the pore assembly or produce reactive sites facing to the pore's exterior instead of to the pore lumen, were also spotted and discussed. All results demonstrated above have provided a complete map of engineering sites within the constriction area of MspA and may be beneficial as a reference in future engineering of corresponding nanoreactors.