Exploring the Aggregation Propensity of PHF6 Peptide Segments of the Tau Protein Using Ion Mobility Mass Spectrometry Techniques.

Peptide and protein aggregation involves the formation of oligomeric species, but the complex interplay between oligomers of different conformations and sizes complicates their structural elucidation. Using ion mobility mass spectrometry (IM-MS), we aim to reveal these early steps of aggregation for the Ac-PHF6-NH2 peptide segment from tau protein, thereby distinguishing between different oligomeric species and gaining an understanding of the aggregation pathway. An important factor that is often neglected, but which can alter the aggregation propensity of peptides, is the terminal capping groups. Here, we demonstrate the use of IM-MS to probe the early stages of aggregate formation of Ac-PHF6-NH2, Ac-PHF6, PHF6-NH2, and uncapped PHF6 peptide segments. The aggregation propensity of the four PHF6 segments is confirmed using thioflavin T fluorescence assays and transmission electron microscopy. A novel approach based on post-IM fragmentation and quadrupole selection on the TIMS-Qq-ToF (trapped ion mobility) spectrometer was developed to enhance oligomer assignment, especially for the higher-order aggregates. This approach pushes the limits of IM identification of isobaric species, whose signatures appear closer to each other with increasing oligomer size, and provides new insights into the interpretation of IM-MS data. In addition, TIMS collision cross section values are compared with traveling wave ion mobility (TWIMS) data to evaluate potential instrumental bias in the trapped ion mobility results. The two IM-MS instrumental platforms are based on different ion mobility principles and have different configurations, thereby providing us with valuable insight into the preservation of weakly bound biomolecular complexes such as peptide aggregates.

Covalent bicyclization of protein complexes yields durable quaternary structures.

Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.

Developments in Trapped Ion Mobility Mass Spectrometry to Probe the Early Stages of Peptide Aggregation.

Ion mobility mass spectrometry (IM-MS) has proven to be an excellent method to characterize the structure of amyloidogenic protein and peptide aggregates, which are formed in coincidence with the development of neurodegenerative diseases. However, it remains a challenge to obtain detailed structural information on all conformational intermediates, originating from the early onset of those pathologies, due to their complex and heterogeneous environment. One way to enhance the insights and the identification of these early stage oligomers is by employing high resolution ion mobility mass spectrometry experiments. This would allow us to enhance the mobility separation and MS characterization. Trapped ion mobility spectrometry (TIMS) is an ion mobility technique known for its inherently high resolution and has successfully been applied to the analysis of protein conformations among others. To obtain conformational information on fragile peptide aggregates, the instrumental parameters of the TIMS-Quadrupole-Time-of-Flight mass spectrometer (TIMS-qToF-MS) have to be optimized to allow the study of intact aggregates and ensure their transmission toward the detector. Here, we investigate the suitability and application of TIMS to probe the aggregation process, targeting the well-characterized M307-N319 peptide segment of the TDP-43 protein, which is involved in the development of amyotrophic lateral sclerosis. By studying the influence of key parameters over the full mass spectrometer, such as source temperature, applied voltages or RFs among others, we demonstrate that by using an optimized instrumental method TIMS can be used to probe peptide aggregation.

Polycyclic aromatic hydrocarbon growth in a benzene discharge explored by IR-UV action spectroscopy.

Infrared signatures of polycyclic aromatic hydrocarbons (PAHs) are detected towards many phases of stellar evolution. PAHs are major players in the carbon chemistry of the interstellar medium, forming the connection between small hydrocarbons and large fullerenes. However, as details on the formation of PAHs in these environments are still unclear, modeling their abundance and chemistry has remained far from trivial. By combining molecular beam mass-selective IR spectroscopy and calculated IR spectra, we analyze the discharge of benzene and identify resulting products including larger PAHs, radicals and intermediates that serve as promising candidates for radio astronomical searches. The identification of various reaction products indicates that different gas-phase reaction mechanisms leading to PAH growth must occur under the same conditions to account for all observed PAH-related species, thereby revealing the complex and interconnected network of PAH formation pathways. The results of this study highlight key (exothermic) reactions that need to be included in astrochemical models describing the carbon chemistry in our universe.

Gas-Phase Infrared Spectra of the C7H5 Radical and Its Bimolecular Reaction Products.

Resonance-stabilized radicals are considered as possible intermediates in the formation of polycyclic aromatic hydrocarbons (PAHs) in interstellar space. Here, we investigate the fulvenallenyl radical, the most stable C7H5 isomer by IR/UV ion dip spectroscopy employing free electron laser radiation in the mid-infrared region between 550 and 1750 cm-1. The radical is generated by pyrolysis from phthalide. Various jet-cooled reaction products are identified by their mass-selective IR spectra in the fingerprint region, based on a comparison with computed spectra. Interestingly, benzyl is present as a second resonance-stabilized radical. It is connected to fulvenallenyl by a sequence of two H atom losses or additions. Among the identified aromatic hydrocarbons are toluene and styrene, as well as polycyclic molecules, such as indene, naphthalene, fluorene and phenanthrene. Mechanisms for the formation of PAH from C7H5 have already been suggested in previous computational work. In particular, the radical/radical reaction of two fulvenallenyl radicals provides an efficient route to phenanthrene in one bimolecular step and might be relevant for PAH formation under astrochemical conditions.

Structural Properties of Phenylalanine-Based Dimers Revealed Using IR Action Spectroscopy.

Peptide segments with phenylalanine residues are commonly found in proteins that are related to neurodegenerative diseases. However, the self-assembly of phenylalanine-based peptides can be also functional. Peptides containing phenylalanine residues with different side caps, composition, and chemical alteration can form different types of nanostructures that find many applications in technology and medicine. Various studies have been performed in order to explain the remarkable stability of the resulting nanostructures. Here, we study the early stages of self-assembly of two phenylalanine derived peptides in the gas phase using IR action spectroscopy. Our focus lies on the identification of the key intra- and intermolecular interactions that govern the formation of the dimers. The far-IR region allowed us to distinguish between structural families and to assign the 2-(2-amino-2-phenylacetamido)-2-phenylacetic acid (PhgPhg) dimer to a very symmetric structure with two intermolecular hydrogen bonds and its aromatic rings folded away from the backbone. By comparison with the phenylalanine-based peptide cyclic L-phenylalanyl-L-phenylalanine (cyclo-FF), we found that the linear FF dimer likely adopts a less ordered structure. However, when one more phenylalanine residue is added (FFF), a more structurally organized dimer is formed with several intermolecular hydrogen bonds.

The gas-phase infrared spectra of the 2-methylallyl radical and its high-temperature reaction products.

The resonance-stabilized 2-methylallyl radical, 2-MA, is considered as a possible intermediate in the formation of polycyclic aromatic hydrocarbons (PAHs) in combustion processes. In this work, we report on its contribution to molecular growth in a high-temperature microreactor and provide mass-selective IR/UV ion dip spectra of the radical, as well as the various jet-cooled reaction products, employing free electron laser radiation in the mid-infrared region. Small (aromatic) hydrocarbons such as fulvene, benzene, styrene, or para-xylene, as well as polycyclic molecules, like (methylated) naphthalene, were identified with the aid of ab initio DFT computations. Several reaction products differ by one or more methyl groups, suggesting that molecular growth is dominated by (de)methylation in the reactor.

Gas-Phase Infrared Spectroscopy of Neutral Peptides: Insights from the Far-IR and THz Domain.

Gas-phase, double resonance IR spectroscopy has proven to be an excellent approach to obtain structural information on peptides ranging from single amino acids to large peptides and peptide clusters. In this review, we discuss the state-of-the-art of infrared action spectroscopy of peptides in the far-IR and THz regime. An introduction to the field of far-IR spectroscopy is given, thereby highlighting the opportunities that are provided for gas-phase research on neutral peptides. Current experimental methods, including spectroscopic schemes, have been reviewed. Structural information from the experimental far-IR spectra can be obtained with the help of suitable theoretical approaches such as dynamical DFT techniques and the recently developed Graph Theory. The aim of this review is to underline how the synergy between far-IR spectroscopy and theory can provide an unprecedented picture of the structure of neutral biomolecules in the gas phase. The far-IR signatures of the discussed studies are summarized in a far-IR map, in order to gain insight into the origin of the far-IR localized and delocalized motions present in peptides and where they can be found in the electromagnetic spectrum.

The Gas-Phase Infrared Spectra of Xylyl Radicals.

The three isomers of the xylyl radical, C8H9, are possible intermediates in the formation of soot and polycyclic aromatic hydrocarbons (PAH). Their infrared spectra have been recorded by IR/UV ion dip spectroscopy using free electron laser radiation. The radicals were generated by flash pyrolysis from the corresponding nitrites and resonantly ionized via the D3 ← D0 transition around 310 nm. Mid-infrared spectra of the three xylyl isomers were recorded between 550 and 1700 cm-1 and are in excellent agreement with computations, provided that overtones and combination bands are included in the simulation. The results show that the three xylyl isomers can be distinguished by their infrared spectra and that no isomerization occurs in the pyrolysis reactor. The IR spectra obtained at m/z = 208 indicate that dimerization of xylyl radicals leads to substituted stilbenes, which has not been observed for benzyl.

Interactions of aggregating peptides probed by IR-UV action spectroscopy.

Peptide aggregation, the self-assembly of peptides into structured beta-sheet fibril structures, is driven by a combination of intra- and intermolecular interactions. Here, the interplay between intramolecular and formed inter-sheet hydrogen bonds and the effect of dispersion interactions on the formation of neutral, isolated, peptide dimers is studied using infrared action spectroscopy. Therefore, four different homo- and heterogenous dimers resulting from three different alanine-based model peptides have been formed under controlled and isolated conditions. The peptides differ from one another by the presence and location of a UV chromophore containing end cap. The conformations of the monomers of the peptides direct the final dimer structure: strongly bonded or folded structures result in weakly bound dimers. Here, intramolecular hydrogen bonds are favored over new intermolecular hydrogen bond interactions. In contrast, linear monomers are the ideal template to form parallel beta-sheet type structures. The weak intramolecular hydrogen bonds present in the linear monomers are replaced by the stronger inter-sheet hydrogen bond interactions. The influence of π-π dispersion interactions on the structure of the dimers is minimal, and the phenyl rings have a tendency to fold away from the peptide backbone to favour intermolecular hydrogen bond interactions over dispersion interactions. Quantum chemical calculations confirm our experimental observations.

Formation of Neutral Peptide Aggregates as Studied by Mass-Selective IR Action Spectroscopy.

The spontaneous aggregation of proteins and peptides is widely studied owing to its relation to neurodegenerative diseases. To understand the underlying principles of peptide aggregation, elucidation of structure and structural changes upon their formation is key. This level of detail can be obtained by studying the peptide self-assembly in the gas phase. Structural characterization of aggregates is mainly done on charged species, as adding charges is an intrinsic part of the technique to bring molecules into the gas phase. Studying neutral peptide aggregates will complement the existing picture. These studies are restricted to dimers due to experimental limitations. Herein, we present advances in laser desorption molecular beam spectroscopy to form neutral peptide aggregates consisting of up to 14 monomeric peptides in the gas phase. The combination of this technique with IR-UV spectroscopy allowed us to select each aggregate by size and subsequently characterize its structure.

Conformational assignment of gas phase peptides and their H-bonded complexes using far-IR/THz: IR-UV ion dip experiment, DFT-MD spectroscopy, and graph theory for mode assignment.

The combined approach of gas phase IR-UV ion dip spectroscopy experiments and DFT-based molecular dynamics simulations for theoretical spectroscopy reveals the 3D structures of (Ac-Phe-OMe)1,2 peptides using their far-IR/THz signatures. Both experimental and simulated IR spectra are well-resolved in the 100-800 cm-1 domain, allowing an unambiguous assignment of the conformers, that could not be achieved in other more congested spectral domains. We also present and make proofs-of-principles for our newly developed theoretical method for the assignment of (anharmonic) vibrational modes from MD simulations based on graph theory coupled to APT-weighted internal coordinates velocities DOS spectra. The principles of the method are reviewed, applications to the simple gas phase water and NMA (N-methyl-acetamide) molecules are presented, and application to the more complex (Ac-Phe-OMe)1,2 peptidic systems shows that the complexity in assigning vibrational modes from MD simulations is reduced with the graphs. Our newly developed graph-based methodology is furthermore shown to allow an easy comparison between the vibrational modes of isolated monomer(s) and their complexes, as illustrated by the (Ac-Phe-OMe)1,2 peptides.

Conformational Preferences of Isolated Glycylglycine (Gly-Gly) Investigated with IRMPD-VUV Action Spectroscopy and Advanced Computational Approaches.

In this article, we report the results of gas-phase IR spectroscopy of neutral glycylglycine (Gly-Gly) in the 700-1850 cm-1 frequency range. A combination of laser desorption, jet-cooling, and IR multiple-photon dissociation vacuum-ultraviolet (IRMPD-VUV) action spectroscopy is employed, together with extensive quantum chemical calculations that assist in the analysis of the experimental data. As a result, we determined that the most favorable conformer in the low-temperature environment of the supersonic jet is the nearly planar structure with two C5 hydrogen-bonding interactions. Calculations clearly show that this conformer is favored because of its flexibility (considerable entropy stabilization) as well as efficient conformer relaxation processes in the jet. To gain more understanding into the relative stability of the lowest-energy Gly-Gly conformers, the relative strength of hydrogen bonding and steric interactions is analyzed using the noncovalent interactions (NCI) approach.

Structural characterization of nucleotide 5'-triphosphates by infrared ion spectroscopy and theoretical studies.

The molecular family of nucleotide triphosphates (NTPs), with adenosine 5'-triphosphate (ATP) as its best-known member, is of high biochemical importance as their phosphodiester bonds form Nature's main means to store and transport energy. Here, gas-phase IR spectroscopic studies and supporting theoretical studies have been performed on adenosine 5'-triphosphate, cytosine 5'-triphosphate and guanosine 5'-triphosphate to elucidate the intrinsic structural properties of NTPs, focusing on the influence of the nucleobase and the extent of deprotonation. Mass spectrometric studies involving collision induced dissociation showed similar fragmentation channels for the three studied NTPs within a selected charge state. The doubly charged anions exhibit fragmentation similar to the energy-releasing hydrolysis reaction in nature, while the singly charged anions show different dominant fragmentation channels, suggesting that the charge state plays a significant role in the favorability of the hydrolysis reaction. A combination of infrared ion spectroscopy and quantum-chemical computations indicates that the singly charged anions of all NTPs are preferentially deprotonated at their ß-phosphates, while the doubly-charged anions are dominantly αß-deprotonated. The assigned three-dimensional structure differs for ATP and CTP on the one hand and GTP on the other, in the sense that ATP and CTP show no interaction between nucleobase and phosphate tail, while in GTP they are hydrogen bonded. This can be rationalized by considering the structure and geometry of the NTPs where the final three dimensional structure depends on a subtle balance between hydrogen bond strength, flexibility and steric hindrance.

Mapping gas phase dipeptide motions in the far-infrared and terahertz domain.

Vibrational signatures of Ac-Phe-AA-NH2 dipeptides are recorded and analysed in the far IR/THz spectral domain (100-800 cm-1, 3-24 THz), with the 'AA' amino acid chosen within the series 'AA' = Gly, Ala, Pro, Cys, Ser, Val. Phe stands for phenylalanine. IR-UV ion dip experiments are conducted on the free electron laser FELIX and combined with DFT-based molecular dynamics simulations for the calculation of the dynamical anharmonic vibrational spectra. The excellent agreements between the experimental and theoretical spectra of the Ac-Phe-AA-NH2 series allow us to make detailed and unambiguous mapping of the vibrational motions into three main domains: 700-800 cm-1 for C-H waggings, 400-700 cm-1 for N-H waggings, with a one-to-one signature per amide N-H backbone group, 0-400 cm-1 for delocalized and large amplitude collective motions over the dipeptide backbone, with backbone torsional motions arising <100 cm-1.

Infrared Action Spectroscopy of Low-Temperature Neutral Gas-Phase Molecules of Arbitrary Structure.

We demonstrate a technique for IR action spectroscopy that enables measuring IR spectra in a background-free fashion for low-temperature neutral gas-phase molecules of arbitrary structure. The method is exemplified experimentally for N-methylacetamide molecules in the mid-IR spectral range of 1000-1800 cm^{-1}, utilizing the free electron laser FELIX. The technique involves the resonant absorption of multiple mid-IR photons, which induces molecular dissociation. The dissociation products are probed with 10.49 eV vacuum ultraviolet photons and analyzed with a mass spectrometer. We also demonstrate the capability of this method to record, with unprecedented ease, mid-IR spectra for the molecular associates, such as clusters and oligomers, present in a molecular beam. In this way the mass-selected spectra of low-temperature gas-phase dimers and trimers of N-methylacetamide are measured in the full amide I-III range.

Far-infrared amide IV-VI spectroscopy of isolated 2- and 4-Methylacetanilide.

Delocalized molecular vibrations in the far-infrared and THz ranges are highly sensitive to the molecular structure, as well as to intra- and inter-molecular interactions. Thus, spectroscopic studies of biomolecular structures can greatly benefit from an extension of the conventional mid-infrared to the far-infrared wavelength range. In this work, the conformer-specific gas-phase far-infrared spectra of two aromatic molecules containing the peptide -CO-NH- link, namely, 2- and 4-Methylacetanilide, are investigated. The planar conformations with trans configuration of the peptide link have only been observed in the supersonic-jet expansion. The corresponding far-infrared signatures associated with the vibrations of the peptide -CO-NH- moiety, the so-called amide IV-VI bands, have been assigned and compared with the results of density functional theory frequency calculations based on the anharmonic vibrational second-order perturbation theory approach. The analysis of the experimental and theoretical data shows that the amide IV-VI bands are highly diagnostic for the geometry of the peptide moiety and the molecular backbone. They are also strongly blue-shifted upon formation of the NH⋯O-C hydrogen bonding, which is, for example, responsible for the formation of secondary protein structures. Furthermore, the amide IV-VI bands are also diagnostic for the cis configuration of the peptide link, which can be present in cyclic peptides. The experimental gas-phase data presented in this work can assist the vibrational assignment of similar biologically important systems, either isolated or in natural environments.

A conformation-selective IR-UV study of the dipeptides Ac-Phe-Ser-NH2 and Ac-Phe-Cys-NH2: probing the SH···O and OH···O hydrogen bond interactions.

The conformational preferences of peptides are mainly controlled by the stabilizing effect of intramolecular interactions. In peptides with polar side chains, not only the backbone but also the side chain interactions determine the resulting conformations. In this paper, the conformational preferences of the capped dipeptides Ac-Phe-Ser-NH2 (FS) and Ac-Phe-Cys-NH2 (FC) are resolved under laser-desorbed jet cooling conditions using IR-UV ion dip spectroscopy and density functional theory (DFT) quantum chemistry calculations. As serine (Ser) and cysteine (Cys) only differ in an OH (Ser) or SH (Cys) moiety; this subtle alteration allows us to study the effect of the difference in hydrogen bonding for an OH and SH group in detail, and its effect on the secondary structure. IR absorption spectra are recorded in the NH stretching region (3200-3600 cm(-1)). In combination with quantum chemical calculations the spectra provide a direct view of intramolecular interactions. Here, we show that both FS as FC share a singly γ-folded backbone conformation as the most stable conformer. The hydrogen bond strength of OH···O (FS) is stronger than that of SH···O (FC), resulting in a more compact gamma turn structure. A second conformer is found for FC, showing a ß turn interaction.

Gas-phase peptide structures unraveled by far-IR spectroscopy: combining IR-UV ion-dip experiments with Born-Oppenheimer molecular dynamics simulations.

Vibrational spectroscopy provides an important probe of the three-dimensional structures of peptides. With increasing size, these IR spectra become very complex and to extract structural information, comparison with theoretical spectra is essential. Harmonic DFT calculations have become a common workhorse for predicting vibrational frequencies of small neutral and ionized gaseous peptides. Although the far-IR region (<500 cm(-1)) may contain a wealth of structural information, as recognized in condensed phase studies, DFT often performs poorly in predicting the far-IR spectra of peptides. Here, Born-Oppenheimer molecular dynamics (BOMD) is applied to predict the far-IR signatures of two γ-turn peptides. Combining experiments and simulations, far-IR spectra can provide structural information on gas-phase peptides superior to that extracted from mid-IR and amide A features.

Gas-phase salt bridge interactions between glutamic acid and arginine.

The gas-phase side chain-side chain (SC-SC) interaction and possible proton transfer between glutamic acid (Glu) and arginine (Arg) residues are studied under low-temperature conditions in an overall neutral peptide. Conformation-specific IR spectra, obtained with the free electron laser FELIX, in combination with density functional theory (DFT) calculations, provide insight into the occurrence of intramolecular proton transfer and detailed information on the conformational preferences of the peptides Z-Glu-Alan-Arg-NHMe (n = 0,1,3). Low-energy structures are obtained using molecular dynamics simulations via the simulated annealing approach, resulting in three types of SC-SC interactions, in particular two types of pair-wise interactions and one bifurcated interaction. These low-energy structures are optimized and frequency calculations are performed using the B3LYP functional, for structural analysis, and the M05-2x functional, for relative energies, employing the 6-311+G(d,p) basis set. Comparison of experimental and computed spectra suggests that only a single conformation was present for each of the three peptides. Despite the increasing spacing between the Glu and Arg residues, the peptides have several types of interactions in common, in particular specific SC-SC and dispersion interactions between the Arg side chain and the phenyl ring of the Z-cap. Comparison with previous experiments on Ac-Glu-Ala-Phe-Ala-Arg-NHMe as well as molecular dynamics simulations further suggest that the pairwise interaction observed here is indeed energetically most favorable for short peptide sequences.