Site-Specific Characterization of Intermediates in Folding-Tetramerization of Melittin by the Rapid Mix/Freeze Method and Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP) NMR at Low Temperature (25K)

Abstract

Protein folding study has been regarded very important for fundamental understanding of protein structures and functions. Although significant progresses have been made, the characterization of the kinetic transition pathways and intermediate states has been challenged and limited by either time or spatial resolutions. In this presentation, I will introduce a newly developed rapid mixing/freezing method for the sensitivity-enhanced solid-state NMR study at low temperature (25 K) by using the dynamic nuclear polarization (DNP) method. Our rapid mixer/freezer device can mix two solutions to introduce the folding within a couple of milliseconds and rapidly freeze at liquid nitrogen temperature (80K) within few microseconds to trap the intermediate states during the folding transition. In this study, the folding and tetramerization of a small bee venom peptide, melittin was investigated. Melittin, selectively isotopically 13C and 15N labeled to monitor the folding and the possible dimerization, underwent rapid pH change and was freeze-trapped at different time points (2, 4, 9, 30 ms). The low temperature MAS-DNP NMR spectra of frozen samples captured at varying transition time points revealed the site-specific changes of the simultaneous folding and oligomeric transitions (monomer-dimer-tetramer). The detail information of the mixer/freezer design, and the characterization of melittin intermediates and its pathway will be discussed in the presentation.