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MAS-DNP NMR investigation of the transient intermediate state of melittin by the rapidly mixing and freezing technique

Thursday, September 14, 2017 — Poster Session III

12:00 p.m. – 1:30 p.m.
FAES Terrace


  • J Jeon
  • KR Thurber
  • YM Yau
  • R Tycko


Characterization of intermediate states in protein folding can reveal the folding pathway precluding otherwise the astronomical number of possible folding pathways. However, the transient character and the low sensitivity from the non-equilibrium species in the measurement of intermediates make the characterization very challenging. In this study, we have developed the microfluidic rapid mixer and rapid freezer system for capturing the intermediate states within several milliseconds, and enhanced the NMR signals, for probing such states, by applying the sensitivity enhancing technique, Dynamic Nuclear Polarization (DNP) solid-state NMR (SSNMR) at ultra-low temperature (25 K). Especially, with this state-of-the-art DNP technique, two-dimensional SSNMR measurements on proteins in lowly populated states of proteins are enabled in 1-4 hours, while otherwise several days of measurement time would be required to achieve adequate signal-to-noise. Melittin is a small peptide known to disrupt cell membrane by creating pores at the surface. Melittin and the modulation of its tetramerization have been investigated for its antimicrobial applications, however, the pathway of the rapid transition between the random coil monomer and the helical tetramer is not understood for these applications. In this study, its conformational changes during folding/oligomerization transition are captured by diluting rapidly the denaturant and freeze-trapping at various time points, and probed in terms of intra- and intermolecular dipolar recoupling distances with different spin diffusion mixing time in SSNMR spectra. In the poster, further discussions on intermediates and folding pathways of melittin will be discussed in conjunction with static and stopped flow circular dichroism and fluorescence spectroscopic analyses.

Category: Biomedical Engineering and Biophysics