Application of millisecond time-resolved solid state NMR to the kinetics and mechanism of melittin self-assembly

Proceedings of the National Academy of Sciences of the United States of America
Jaekyun JeonRobert Tycko

Abstract

Common experimental approaches for characterizing structural conversion processes such as protein folding and self-assembly do not report on all aspects of the evolution from an initial state to the final state. Here, we demonstrate an approach that is based on rapid mixing, freeze-trapping, and low-temperature solid-state NMR (ssNMR) with signal enhancements from dynamic nuclear polarization (DNP). Experiments on the folding and tetramerization of the 26-residue peptide melittin following a rapid pH jump show that multiple aspects of molecular structure can be followed with millisecond time resolution, including secondary structure at specific isotopically labeled sites, intramolecular and intermolecular contacts between specific pairs of labeled residues, and overall structural order. DNP-enhanced ssNMR data reveal that conversion of conformationally disordered melittin monomers at low pH to α-helical conformations at neutral pH occurs on nearly the same timescale as formation of antiparallel melittin dimers, about 6 to 9 ms for 0.3 mM melittin at 24 °C in aqueous solution containing 20% (vol/vol) glycerol and 75 mM sodium phosphate. Although stopped-flow fluorescence data suggest that melittin tetramers form quickly after di...Continue Reading

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Citations

Sep 2, 2020·Biomolecules·Nhi T TranJoanna R Long
May 10, 2020·Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry·Thomas SchmidtG Marius Clore
Feb 18, 2021·Proceedings of the National Academy of Sciences of the United States of America·Xiaoming ZhouSteven L McKnight
Apr 30, 2021·Current Opinion in Structural Biology·Marta BonaccorsiGuido Pintacuda
May 1, 2021·Molecular Immunology·Carlos Leónidas LeivaPablo Chacana
Aug 10, 2021·Nature Reviews. Methods Primers·Bernd ReifMei Hong
Jun 26, 2020·Langmuir : the ACS Journal of Surfaces and Colloids·Remi Casier, Jean Duhamel

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