From Solution to the Gas Phase: Factors That Influence Kinetic Trapping of Substance P in the Gas Phase.

Substance P (RPKPQQFFGLM-NH2) [M + 3H]3+ ions have been shown to exist as two conformers: a kinetically-trapped conformer and a thermodynamically more stable, and therefore energetically preferred conformer. Molecular dynamics (MD) simulations suggested that the kinetically trapped population is stabilized by interactions between the charge sites and the polar side chains of glutamine (Q) located at positions 5 and 6 and phenylalanine (F) located at positions 7 and 8. Here, the individual contributions of these specific intramolecular interactions are systematically probed through site-directed alanine mutations of the native amino acid sequence. Ion mobility spectrometry (IMS) data for the mutant peptide ions confirm that interactions between the charge sites and glutamine/phenylalanine side chains afford stabilization of the kinetically trapped ion population. In addition, experimental data for proline-to-alanine mutations at positions 2 and 4 clearly show that interactions involving the charge sites and the Q/F side chains are altered by cis-/trans- orientations of the proline residues, and the mutation glycine-to-proline at position 9 supports results from MD simulations that suggest the C-terminus also provides stabilization of the kinetically-trapped conformation.