Size-Selective VAILase Proteolysis Provides Dynamic Insights into Protein Structures

Monitoring the dynamic alterations
of protein structures within
an aqueous solution remains enormously challenging. In this study,
we describe a size-selective VAILase proteolysis (SVP)-mass spectrometry
(MS) strategy to probe the protein structure changes without strict
control of the proteolysis kinetics. The unique conformation selectivity
of SVP depends on the uniform nano-sized entrance pores of the VAILase
hexameric cage as well as the six inherent molecular rulers in the
VAILase–substrate recognition and cleavage. The dynamic insights
into subtle conformation alterations of both myoglobin unfolding transition
and Aurora kinase A-inhibitor binding are successfully captured using
the SVP strategy, which matches well with the results in the molecular
dynamics simulation. Our work provides a new paradigm of size-selective
native proteolysis for exploring the aqueous protein structure–function
relationships.