High Pressure CPMG and CEST Reveal That Cavity Position Dictates Distinct Dynamic Disorder in the PP32 Repeat Protein

Area of Science:

• Protein dynamics
• Biophysics
• Structural biology

Background:

• Protein conformational dynamics are crucial for function.
• High pressure (HP) perturbation aids in studying excited states with smaller volumes.
• Repeat proteins serve as models for sequence-dependent dynamics.

Purpose of the Study:

• To characterize time scales and structures of protein conformational transitions.
• To investigate the role of sequence in protein conformational dynamics using HP perturbation.
• To probe the effect of cavity introduction on protein dynamics.

Main Methods:

• ¹⁵N CPMG relaxation dispersion analysis.
• ¹⁵N-CEST (Chemical Exchange Saturation Transfer) analysis.
• High pressure (HP) perturbation techniques.

Main Results:

• A cavity mutation (I7A) in pp32 protein induced pressure-dependent conformational exchange on the 500 μs-2 ms time scale.
• Exchange amplitude showed a gradient from N- to C-terminus.
• A different mutation (L60A) in the pp32 core induced slower pressure-induced exchange (>2 ms).
• HP CEST revealed distinct structural changes: N-terminus unfolded, core remained native-like in the excited state.

Conclusions:

• Cavity position is critical in determining the time scales of conformational exchange.
• Sequence subtly yet centrally dictates protein conformational dynamics.
• HP chemical exchange measurements provide insights into protein flexibility and function.