Temperature-Dependent\nTrimethylamine <i>N</i>‑Oxide Induced the Formation\nof Substance P Dimers

Interactions of the peptide substance P (SP) (RPKPQQFFGLM-NH<sub>2</sub>) with trimethylamine <i>N</i>-oxide (TMAO) were\ninvestigated by using cryo-ion mobility-mass spectrometry (cryo-IM-MS),\nvariable-temperature (278–358 K) electrospray ionization (vT-ESI) MS, and\nmolecular dynamics (MD) simulations. Cryo-IM-MS provides evidence\nthat cold solutions containing SP and TMAO yield abundant hydrated\nSP dimer ions, but dimer formation is inhibited in solutions that\nalso contain urea. In addition, we show that SP dimer formation at\ncold solution temperatures (<298 K) is favored when TMAO interacts\nwith the hydrophobic C-terminus of SP and is subject to reduced entropic\npenalty when compared to warmer solution conditions (>298 K). MD\nsimulations\nshow that TMAO lowers the free energy barrier for dimerization and\nthat monomers dimerize by forming hydrogen bonds (HBs). Moreover,\ndifferences in oligomer abundances for SP mutants (P4A, P2,4A, G9P,\nand P2,4A/G9P) provide evidence that oligomerization facilitated by\nTMAO is sensitive to the <i>cis</i>/<i>trans</i> orientation of residues at positions 2, 4, and 9.