Reaching the protein folding speed limit with large, sub-microsecond pressure jumps

Area of Science:

• Biophysics
• Protein dynamics
• Biomolecular engineering

Background:

• Biomolecules exhibit high pressure sensitivity, but their rapid dynamics upon pressure release are difficult to study.
• Existing methods lack the speed and sample efficiency to capture these fast processes.

Purpose of the Study:

• To develop a novel, sample-efficient method for inducing large and rapid pressure drops.
• To investigate the fast refolding dynamics of biomolecules after pressure-induced denaturation.

Main Methods:

• Developed a method enabling pressure drops greater than 2,500 atmospheres in under 0.7 microseconds with low sample consumption (<1 nanomole).
• Utilized fluorescence-detected refolding of a genetically engineered lambda repressor mutant to validate the technique.

Main Results:

• Successfully resolved barrierless structure formation in a lambda repressor mutant upon return to ambient pressure.
• Measured a refolding time of 2.1 ± 0.7 microseconds, approaching the theoretical speed limit for protein folding.
• Observed significantly faster refolding compared to temperature-jump methods for the same protein.

Conclusions:

• The new method allows experimental access to previously unobservable, ultrafast biomolecular dynamics.
• This technique provides a powerful tool for exploring the biomolecular energy landscape at an atomic level.
• Opens new avenues for simulating and understanding protein folding and other pressure-sensitive processes.