Size-Selective VAILase Proteolysis Provides Dynamic Insights into Protein Structures.

Area of Science:

• Biochemistry
• Structural Biology
• Analytical Chemistry

Background:

• Monitoring dynamic protein structure changes in aqueous solutions is a significant analytical challenge.
• Existing methods may require strict control over proteolysis kinetics, limiting their applicability.

Purpose of the Study:

• To develop a novel strategy for probing dynamic protein structure alterations in aqueous environments.
• To overcome limitations in monitoring protein conformation changes without precise control of proteolysis kinetics.

Main Methods:

• A size-selective VAILase proteolysis (SVP) coupled with mass spectrometry (MS) strategy was developed.
• The method leverages the unique conformation selectivity of VAILase, utilizing its nano-sized pores and molecular rulers for substrate recognition and cleavage.
• Applied to study myoglobin unfolding and Aurora kinase A-inhibitor binding dynamics.

Main Results:

• The SVP-MS strategy successfully captured dynamic insights into subtle conformation alterations of myoglobin unfolding.
• The binding of Aurora kinase A to its inhibitor was also effectively monitored, revealing dynamic structural changes.
• Results obtained via SVP-MS showed strong agreement with molecular dynamics simulations.

Conclusions:

• The developed size-selective native proteolysis strategy offers a new paradigm for studying aqueous protein structure-function relationships.
• SVP-MS provides dynamic insights into protein conformational changes, complementing traditional simulation methods.
• This technique enhances the ability to explore protein dynamics in biologically relevant solution conditions.