Millisecond Time-Resolved Solid-State NMR Initiated by Rapid Inverse Temperature Jumps

Area of Science:

• Biophysical Chemistry
• Structural Biology
• Biomolecular Dynamics

Background:

• Understanding macromolecular structural conversions (folding, complex formation, self-assembly) is crucial in biophysical chemistry.
• New experimental methods are needed to probe these rapid processes.
• Melittin peptide undergoes a significant structural transition from disordered monomer to α-helical tetramer upon temperature change.

Purpose of the Study:

• To introduce and demonstrate a novel rapid inverse temperature jump technique for initiating and studying biomolecular structural conversions.
• To elucidate the kinetics and mechanism of melittin's structural transition from disordered monomer to α-helical tetramer.

Main Methods:

• Developed a rapid inverse temperature jump (T-jump) method by rapidly cooling solutions (95 °C to 30 °C in ~0.8 ms) using capillary tubes.
• Time-resolved solid-state nuclear magnetic resonance (ssNMR) was employed to analyze structural evolution after the T-jump.
• Solutions were rapidly frozen after variable structural evolution times (τe) and analyzed using ssNMR with dynamic nuclear polarization (DNP) enhancement at 25 K.

Main Results:

• The inverse T-jump technique successfully initiated and allowed time-resolved analysis of melittin's structural conversion.
• ssNMR data showed the development of α-helical secondary structure and intermolecular contacts on a timescale of ~6 ms.
• Kinetic analysis of spectral changes indicated that dimerization is the rate-limiting step in melittin tetramer formation.

Conclusions:

• The rapid inverse T-jump method is effective for studying fast biomolecular structural dynamics.
• Melittin tetramer formation proceeds via a unidirectional dimerization mechanism.
• Dimerization is the slowest step in the overall tetramerization process of melittin.