Misplaced helix slows down ultrafast pressure-jump protein folding.

Area of Science:

• Protein dynamics
• Biophysics
• Structural biology

Background:

• Understanding protein folding is crucial for molecular biology.
• The initial conditions of protein denaturation impact refolding pathways.
• Pressure denaturation offers a unique probe into protein conformational states.

Purpose of the Study:

• To investigate the microsecond to millisecond refolding kinetics of the helix-stabilized protein λ*YA after pressure-induced denaturation.
• To identify the structural basis for slower refolding phases observed after pressure jumps.
• To complement experimental findings with molecular dynamics simulations of protein refolding.

Main Methods:

• Utilized a microsecond pressure-jump apparatus to monitor protein refolding kinetics.
• Performed extensive all-atom molecular dynamics simulations (over 50 μs) of pressure-drop induced refolding.
• Analyzed refolding pathways using multiple force fields to assess structural intermediates.

Main Results:

• Observed both microsecond and a slower 1.4-millisecond refolding phases for λ*YA.
• Pressure denaturation yielded a reversible, helix-coil-rich state, distinct from temperature denaturation.
• Molecular dynamics simulations revealed compact nonnative states with misplaced α-helix content within microseconds.

Conclusions:

• The pressure-denatured state of λ*YA is characterized by residual helix and minimal β-sheet.
• Nonnative helical structures at helix-turn interfaces likely form kinetic traps, delaying refolding.
• Predicted specific mutations to accelerate refolding from the pressure-denatured state, validating the helical trap hypothesis.