Probing the Stability of a ß-Hairpin Scaffold after Desolvation.

Probing the structural characteristics of biomolecular ions in the gas phase following native mass spectrometry (nMS) is of great interest, because noncovalent interactions, and thus native fold features, are believed to be largely retained upon desolvation. However, the conformation usually depends heavily on the charge state of the species investigated. In this study, we combine transition metal ion Förster resonance energy transfer (tmFRET) and ion mobility-mass spectrometry (IM-MS) with molecular dynamics (MD) simulations to interrogate the ß-hairpin structure of GB1p in vacuo. Fluorescence lifetime values and collisional cross sections suggest an unfolding of the ß-hairpin motif for higher charge states. MD simulations are consistent with experimental constraints, yet intriguingly provide an alternative structural interpretation: preservation of the ß-hairpin is not only predicted for 2+ but also for 4+ charged species, which is unexpected given the substantial Coulomb repulsion for small secondary structure scaffolds.

Transition Metal Ion FRET-Based Probe to Study Cu(II)-Mediated Amyloid-ß Ligand Binding.

Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-ß (Αß) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aß complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αß species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aß-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αß, which discloses the binding region and the inhibitor's orientation.

Biochemical basis for the formation of organ-specific volatile blends in mint.

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint (Mentha Ë£ piperita L.), spearmint (Mentha spicata L.) and horsemint (Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs - namely stems, rhizomes and roots - also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae-spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli, purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (-)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (-)-ß-pinene and (-)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs.

Determining the gas-phase structures of α-helical peptides from shape, microsolvation, and intramolecular distance data.

Mass spectrometry is a powerful technique for the structural and functional characterization of biomolecules. However, it remains challenging to accurately gauge the gas-phase structure of biomolecular ions and assess to what extent native-like structures are maintained. Here we propose a synergistic approach which utilizes Förster resonance energy transfer and two types of ion mobility spectrometry (i.e., traveling wave and differential) to provide multiple constraints (i.e., shape and intramolecular distance) for structure-refinement of gas-phase ions. We add microsolvation calculations to assess the interaction sites and energies between the biomolecular ions and gaseous additives. This combined strategy is employed to distinguish conformers and understand the gas-phase structures of two isomeric α-helical peptides that might differ in helicity. Our work allows more stringent structural characterization of biologically relevant molecules (e.g., peptide drugs) and large biomolecular ions than using only a single structural methodology in the gas phase.

The Structure of Cyclic Neuropeptide Somatostatin and Octapeptide Octreotide in the Presence of Copper Ions: Insights from Transition Metal Ion FRET and Native Ion Mobility-Mass Spectrometry.

The conformation and function of somatostatin (SST), a cyclic neuropeptide, was recently found to be altered in the presence of Cu(II) ions, which leads to self-aggregation and loss of biological function as a neurotransmitter. However, the impact of Cu(II) ions on the structure and function of SST is not fully understood. In this work, transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) were utilized to study the structures of well-defined gas-phase ions of SST and of a smaller analogue, octreotide (OCT). The tmFRET results suggest two binding sites of Cu(II) ions in both native-like SST and OCT ions, either in close proximity to the disulfide bond or complexed by two aromatic residues, consistent with results obtained from collision-induced dissociation (CID). The former binding site was reported to initiate aggregation of SST, while the latter binding site could directly affect the essential motif for receptor binding and therefore impair the biological function of SST and OCT when bound to SST receptors. Our results demonstrate that tmFRET is capable of locating transition metal ion binding sites in neuropeptides. Furthermore, multiple distance constraints (tmFRET) and global shape (IM-MS) provide additional structural insights of SST and OCT ions upon metal binding, which is related to the self-aggregation mechanisms and overall biological functions.

Structural Studies of a Stapled Peptide with Native Ion Mobility-Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase.

Native mass spectrometry has emerged as an important tool for gas-phase structural biology. However, the conformations that a biomolecular ion adopts in the gas phase can differ from those found in solution. Herein, we report a synergistic, native ion mobility-mass spectrometry (IM-MS) and transition metal ion Förster resonance energy transfer (tmFRET)-based approach to probe the gas-phase ion structures of a nonstapled peptide (nsp; Ac-CAARAAHAAAHARARA-NH2) and a stapled peptide (sp; Ac-CXARAXHAAAHARARA-NH2). The stapled peptide contains a single hydrocarbon chain connecting the peptide backbone in the i and i + 4 positions via a Grubbs ring-closure metathesis. Fluorescence lifetime measurements indicated that the Cu-bound complexes of carboxyrhodamine 6g (crh6g)-labeled stapled peptide (sp-crh6g) had a shorter donor-acceptor distance (rDA) than the labeled nonstapled peptide (nsp-crh6g). Experimental collision cross-section (CCS) values were then determined by native IM-MS, which could separate the conformations of Cu-bound complexes of nsp-crh6g and sp-crh6g. Finally, the experimental CCS (i.e., shape) and rDA (i.e., distance) values were used as constraints for computational studies, which unambiguously revealed how a staple reduces the elongation of the peptide ions in the gas phase. This study demonstrates the superiority of combining native IM-MS, tmFRET, and computational studies to investigate the structure of biomolecular ions.