Protein hydrogen exchange mechanism: local fluctuations

Area of Science:

• Protein dynamics and structural biology
• Biochemistry and biophysics

Background:

• Protein hydrogen exchange (HX) is a key technique for studying dynamic structural motions.
• Understanding these motions is crucial for elucidating protein function and stability.

Purpose of the Study:

• To investigate the relationship between dynamic structural motions and protein hydrogen exchange (HX) behavior.
• To characterize the impact of a specific mutation (Lys8Gly) on HX rates and protein flexibility.

Main Methods:

• Utilized recombinant cytochrome c with a Lys8Gly mutation to assess effects on protein structure and dynamics.
• Analyzed amide hydrogen exchange rates at various residue locations.
• Compared HX patterns with theoretical models of local fluctuation and concerted unfolding.

Main Results:

• The Lys8Gly mutation increased local main-chain flexibility, accelerating HX of the mutated residue ~50-fold.
• HX changes indicated coupled structural distortions extending several residues but exposing one amide NH at a time.
• Distinguished between 'local fluctuation' HX mode (low destabilant dependence) and 'concerted unfolding' mode (high destabilant sensitivity).

Conclusions:

• Protein hydrogen exchange is governed by distinct dynamic modes, including local fluctuations and global unfolding.
• The Lys8Gly mutation provides a model for studying local HX mechanisms.
• Ionic catalysts access amide hydrogens from bulk solvent in both HX modes.