Structural Landscape of Syanodin I Lasso Peptide

Here, solution NMR, gas-phase hydrogen/deuterium back-exchange (HDbX)-trapped ion mobility spectrometry (TIMS), electron capture dissociation (ECD), quantum mechanical (QM) calculations, and molecular dynamics (MD) simulations were combined for comprehensive structural elucidation of the lasso peptide syanodin I. NMR and ECD MS/MS confirmed an entangled lasso structure with Gln13 as the plug residue maintaining the lasso thread. A maximal c^•/c' ratio at Ala11 (c₁₁^•) was consistent with multiple long-range NOE correlations, identifying Ala11 in proximity to the macrolactam ring. ECD fragmentation patterns indicated a salt bridge between the C-terminus and the Gln13 side chain. TIMS resolved four distinct IMS bands for [M + 2H]²⁺ ions of syanodin I and its branched-cyclic analog over a similar collision cross-sectional range. HDX-MS revealed mass shifts of ∼17 and ∼20 deuteriums for the lasso and branched-cyclic forms, respectively, consistent with the folded nature of the branched-cyclic C-terminal region. HDbX-TIMS-MS experiments (t₀ ∼ 0.72 to t₉ ∼ 865 ms) resolved at least two distinct conformers within each IMS band, revealing intramolecular interactions inaccessible by TIMS alone. QM calculations determined the HDX rate and number of accessible hydrogens for Pro10 and Gln13; this information was used to inform the MD candidate assignment of the 2D-HDX-TIMS-MS results. This workflow provides a comprehensive framework for probing biomolecular conformational dynamics through complementary solution- and gas-phase approaches. The integration of solution-phase hydrogen/deuterium exchange (HDX) with ion mobility spectrometry-mass spectrometry (IMS-MS) offers powerful structural insights into the conformational dynamics of biological molecules.