Hélène Lavanant

Ion Mobility–Mass Spectrometry of Lasso Peptides: Signature of a Rotaxane Topology

Ion mobility mass spectrometry data were collected on a set of five class II lasso peptides and their branched-cyclic topoisomers prepared in denaturing solvent conditions with and without sulfolane as a supercharging agent. Sulfolane was shown not to affect ion mobility results and to allow the formation of highly charged multiply protonated molecules. Drift time values of low charged multiply protonated molecules were found to be similar for the two peptide topologies, indicating the branched-cyclic peptide to be folded in the gas phase into a conformation as compact as the lasso peptide. Conversely, high charge states enabled a discrimination between lasso and branched-cyclic topoisomers, as the former remained compact in the gas phase while the branched-cyclic topoisomer unfolded. Comparison of the ion mobility mass spectrometry data of the lasso and branched-cyclic peptides for all charge states, including the higher charge states obtained with sulfolane, yielded three trends that allowed differentiation of the lasso form from the branched-cyclic topology: low intensity of highly charged protonated molecules, even with the supercharging agent, low change in collision cross sections with increasing charge state of all multiply protonated molecules, and narrow ion mobility peak widths associated with the coexistence of fewer conformations and possible conformational changes.

IRMPD Spectroscopy: Evidence of Hydrogen Bonding in the Gas Phase Conformations of Lasso Peptides and their Branched-Cyclic Topoisomers

Lasso peptides are natural products characterized by a mechanically interlocked topology. The conformation of lasso peptides has been probed in the gas phase using ion mobility-mass spectrometry (IM-MS) which showed differences in the lasso and their unthreaded branched-cyclic topoisomers depending on the ion charge states. To further characterize the evolution of gas phase conformations as a function of the charge state and to assess associated changes in the hydrogen bond network, infrared multiple photon dissociation (IRMPD) action spectroscopy was carried out on two representative lasso peptides, microcin J25 (MccJ25) and capistruin, and their branched-cyclic topoisomers. For the branched-cyclic topoisomers, spectroscopic evidence of a disruption of neutral hydrogen bonds were found when comparing the 3+ and 4+ charge states. In contrast, for the lasso peptides, the IRMPD spectra were found to be similar for the two charge states, suggesting very little difference in gas phase conformations upon addition of a proton. The IRMPD data were thus found consistent and complementary to IM-MS, confirming the stable and compact structure of lasso peptides in the gas phase.

Toward a Rational Design of Highly Folded Peptide Cation Conformations. 3D Gas-Phase Ion Structures and Ion Mobility Characterization

AbstractHeptapeptide ions containing combinations of polar Lys, Arg, and Asp residues with non-polar Leu, Pro, Ala, and Gly residues were designed to study polar effects on gas-phase ion conformations. Doubly and triply charged ions were studied by ion mobility mass spectrometry and electron structure theory using correlated ab initio and density functional theory methods and found to exhibit tightly folded 3D structures in the gas phase. Manipulation of the basic residue positions in LKGPADR, LRGPADK, KLGPADR, and RLGPADK resulted in only minor changes in the ion collision cross sections in helium. Replacement of the Pro residue with Leu resulted in only marginally larger collision cross sections for the doubly and triply charged ions. Disruption of zwitterionic interactions in doubly charged ions was performed by converting the C-terminal and Asp carboxyl groups to methyl esters. This resulted in very minor changes in the collision cross sections of doubly charged ions and even slightly diminished collision cross sections in most triply charged ions. The experimental collision cross sections were related to those calculated for structures of lowest free energy ion conformers that were obtained by extensive search of the conformational space and fully optimized by density functional theory calculations. The predominant factors that affected ion structures and collision cross sections were due to attractive hydrogen bonding interactions and internal solvation of the charged groups that overcompensated their Coulomb repulsion. Structure features typically assigned to the Pro residue and zwitterionic COO-charged group interactions were only secondary in affecting the structures and collision cross sections of these gas-phase peptide ions. Graphical Abstractᅟ

Gas‐phase conformations of capistruin – comparison of lasso, branched‐cyclic and linear topologies

RATIONALE Capistruin is a peptide synthesized by Burkholderia thailandensis E264, which displays a lasso topology. This knot-like structure confers interesting properties to peptides (e.g. antibacterial). Therefore, it is important to evaluate the sensitivity of structural characterization methods to such topological constraints. METHODS Ion mobility mass spectrometry (IMS-MS) experiments, using both drift tube and travelling wave instruments, were performed on lasso capistruin and on peptides with the same sequence, but displaying a branched-cyclic (un-threaded) or linear topology. Molecular dynamics (MD) simulations were then performed to further interpret the IMS results in terms of conformation. RESULTS The collision cross sections (CCSs) measured via IMS for the different forms of capistruin were found to be similar, despite their different topologies for the doubly charged species, but significant differences arise as the charge state is increased. MD simulations for the doubly charged linear peptide were consistent with the hypothesis that salt bridges are present in the gas phase. Moreover, through CCS measurements for peptides with site-specific mutations, the arginine residue at position 11 was found to play a major role in the stabilization of compact structures for the linear peptide. CONCLUSIONS Differences in peptide topologies did not yield marked signatures in their respective IMS spectra. Such signatures were only visible for relatively high charge states, that allow Coulomb repulsion to force unfolding. At low charge states, the topologically unconstrained linear form of capistruin was found to adopt charge solvation-constrained structures, possibly including salt bridges, with CCSs comparable to those measured for the topologically constrained lasso form.

Charge Effect on the Formation of Polyoxometalate-Based Supramolecular Polygons Driven by Metal Coordination

The metal-driven self-assembly of a Keggin-based hybrid bearing two remote pyridine units was investigated. The resulting supramolecular species were identified by combination of 2D diffusion NMR spectroscopy (DOSY) and electrospray ionization mass spectrometry (ESI-MS) as a mixture of molecular triangles and squares. This behavior is different from that of the structural analogue Dawson-based hybrid displaying a higher charge, which only led to the formation of molecular triangles. This study highlights the decisive effect of the charge of the POMs in their self-assembly processes that disfavors the formation of large assemblies. An isothermal titration calorimetry (ITC) experiment confirmed the stronger binding in the case of the Keggin hybrids. A correlation between the diffusion coefficient D and the molecular mass M of the POM-based building block and its coordination oligomers was also observed. We show that the diffusion coefficient of these compounds is mainly determined by their occupied volume rather than by their shape.

Exploration of polyamide structure–property relationships by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

RATIONALE Polyamides (PA) are among the most used classes of polymers because of their attractive properties. Depending on the nature and proportion of the co-monomers used for their synthesis, they can exhibit a very large range of melting temperatures (Tm ). This study aims at the correlation of data from mass spectrometry (MS) with differential scanning calorimetry (DSC) and X-ray diffraction analyses to relate molecular structure to physical properties such as melting temperature, enthalpy change and crystallinity rate. METHODS Six different PA copolymers with molecular weights around 3500 g mol(-1) were synthesized with varying proportions of different co-monomers (amino-acid AB/di-amine AA/di-acid BB). Their melting temperature, enthalpy change and crystallinity rate were measured by DSC and X-ray diffraction. Their structural characterization was carried out by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Because of the poor solubility of PA, a solvent-free sample preparation strategy was used with 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix and sodium iodide as the cationizing agent. RESULTS The different proportions of the repeating unit types led to the formation of PA with melting temperatures ranging from 115°C to 185°C. The structural characterization of these samples by MALDI-TOF-MS revealed a collection of different ion distributions with different sequences of repeating units (AA, BB; AB/AA, BB and AB) in different proportions according to the mixture of monomers used in the synthesis. The relative intensities of these ion distributions were related to sample complexity and structure. They were correlated to DSC and X-ray results, to explain the observed physical properties. CONCLUSIONS The structural information obtained by MALDI-TOF-MS provided a better understanding of the variation of the PA melting temperature and established a structure-properties relationship. This work will allow future PA designs to be monitored.