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Characterization of native structures of proteins in the gas phase remains challenging due to the unpredictable conformational changes the molecules undergo during desolvation and ionization. We spectroscopically studied cryogenically cooled protonated protein ubiquitin and its microhydrated complexes prepared in the gas phase in a range of charge states under different ionization conditions. The UV spectra appear vibrationally resolved for the unfolded protein, but become redshifted and smooth for the native-like structures of ubiquitin. This spectroscopic change results from the H-bonding of the hydroxyl of Tyr to the amide group of Glu-51 in the compact structures; the minimum length of this bond was estimated to be ∼1.7 Å. IR spectroscopy reflects the global structural change by observing redshifts of free NH/OH-stretch vibrational transitions. Evaporative cooling of microhydrated complexes of ubiquitin keeps the protein chilly during ionization, enabling native-like conformers with up to eight protons to survive in the gas phase.

The early stages of fibril formation are difficult to capture in solution. We use cold-ion spectroscopy to examine an 11-residue peptide derived from the protein transthyretin and clusters of this fibre-forming peptide containing up to five units in the gas phase. For each oligomer, the UV spectra exhibit distinct changes in the electronic environment of aromatic residues in this peptide compared to that of the monomer and in the bulk solution. The UV spectra of the tetra- and pentamer are superimposable but differ significantly from the spectra of the monomer and trimer. Such a spectral evolution suggests that a common structural motif is formed as early as the tetramer. The presence of this stable motif is further supported by the low conformational heterogeneity of the tetra- and pentamer, revealed from their IR spectra. From comparison of the IR-spectra in the gas and condensed phases, we propose putative assignments for the dominant motif in the oligomers., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intrinsic structures of biomolecules in the gas phase may not reflect their native solution geometries. Microsolvation of the molecules bridges the two environments, enabling a tracking of molecular structural changes upon hydration at the atomistic level. We employ density functional calculations to compute a large pool of structures and vibrational spectra for a gas-phase complex, in which a doubly protonated decapeptide, gramicidin S, is solvated by two water molecules. Though most vibrations of this large complex are treated in a harmonic approximation, the water molecules and the vibrations of the host ion coupled to them are locally described by a quantum mechanical vibrational self-consistent field theory with second-order perturbation correction (VSCF-PT2). Guided and validated by the available cold ion spectroscopy data, the computational analysis identifies structures of the three experimentally observed conformers of the complex. They, mainly, differ by the hydration sites, of which the one at the Orn side chain is the most important for reshaping the peptide toward its native structure. The study demonstrates the ability of a quantum chemistry approach that intelligently combines the semiempirical and ab initio computations to disentangle a complex interplay of intra- and intermolecular hydrogen bonds in large molecular systems.

We describe the facilities for ultraviolet studies in the femtosecond to nanosecond time domain. These facilities consist of: i) a set-up for deep-ultraviolet spectroscopy in the 260-380 nm range in both pump and probe pulses for transient absorption/reflectivity or two-dimensional spectroscopy studies; ii) a set-up for ultrafast fluorescence measurements with detection down to 300 nm. The capabilities of these set-ups are demonstrated by examples on molecular systems, biosystems, nanoparticles and solid materials.

Because of both experimental and computational challenges, protonated tryptophan has remained the last aromatic amino acid for which the intrinsic structures of low-energy conformers have not been unambiguously solved. The IR-IR-UV hole-burning spectroscopy technique has been applied to overcome the limitations of the commonly used IR-UV double resonance technique and to measure conformer-specific vibrational spectra of TrpH(+), cooled to T = 10 K. Anharmonic ab initio vibrational spectroscopy simulations unambiguously assign the dominant conformers to the two lowest-energy geometries from benchmark coupled-cluster structure computations. The match between experimental and ab initio spectra provides an unbiased validation of the calculated structures of the two experimentally observed conformers of this benchmark ion. Furthermore, the vibrational spectra provide conformer-specific signatures of the stabilizing interactions, including hydrogen bonding and an intramolecular cation-π interaction.

Calculated structures of the two most stable conformers of a protonated decapeptide gramicidin S in the gas phase have been validated by comparing the vibrational spectra, calculated from first- principles and measured in a wide spectral range using infrared (IR)-UV double resonance cold ion spectroscopy. All the 522 vibrational modes of each conformer were calculated quantum mechanically and compared with the experiment without any recourse to an empirical scaling. The study demonstrates that first-principles calculations, when accounting for vibrational anharmonicity, can reproduce high-resolution experimental spectra well enough for validating structures of molecules as large as of 200 atoms. The validated accurate structures of the peptide may serve as templates for in silico drug design and absolute calibration of ion mobility measurements., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The accurate and unambiguous detection of post-translational modifications in proteins and peptides remains a challenging task. We report here the use of cold ion spectroscopy for the identification of phosphorylated tyrosine residues in peptides. This approach employs the wavelength-specific UV fragmentation of cryogenically cooled protonated peptides in the gas phase. In addition to the appearance of specific photofragments, the phosphorylation of tyrosine induces large spectral shifts of the peptide electronic band origins. Quantum chemical calculations and experiments together suggest a certain generality of the use of such shifts in the spectroscopic identification of phosphotyrosines. The enhanced selectivity offered by the joint application of wavelength-specific fragmentation and mass spectrometry of cold molecules can also be used in the identifications of aromatic residues in protonated peptides and, potentially, of other UV-absorbing groups in a variety of large polyatomic ions.

Spectroscopic fingerprint: Infrared–ultraviolet double resonance photodissociation is used for conformational assignment of the electronic spectra of a cold protonated decapeptide (see picture). A mechanism of the IR–UV depletion spectroscopy is proposed and a procedure of using it for measurements of absolute absorption cross-sections of vibrational transitions is elaborated.

Studying solvation of a large molecule on an atomic level is challenging because of the transient character and inhomogeneity of hydrogen bonding in liquid water. We studied water clusters of a protonated macrocyclic decapeptide, gramicidin S, which were prepared in the gas phase and then cooled to cryogenic temperatures. The experiment spectroscopically tracked fine structural changes of the clusters upon increasing the number of attached water molecules from 1 to 50 and distinguished vibrational fingerprints of different conformers. The data indicate that only the first two water molecules induce a substantial change of the gramicidin S structure by breaking two intramolecular noncovalent bonds. The peptide structure remains largely intact upon further solvation, reflecting the interplay between the strong intramolecular and weaker intermolecular hydrogen bonds.

Dimethylaminonitrene complexes of IMesM(+) (IMes =1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene; M = Cu, Ag, Au) were prepared in the gas phase and structurally characterized by high-resolution infrared spectroscopy of the cold species, ion-molecule reactions, and DFT computations. We measured the binding energies of the nitrene fragment to the IMesM(+) moiety by energy-resolved collision-induced dissociation experiments in the gas phase, affording a trend in bond strength of M = Cu ≈ Au > Ag. This trend is explained in terms of a detailed metal-nitrogen bonding analysis, from which relativistic effects on the bonding were assessed. Various density functionals were evaluated for reproducing the observed thermochemical data and Truhlar's M06 functional was found to give the best agreement.

We have measured a vibrationally resolved UV spectrum of doubly protonated gramicidin S (GS) in the gas phase and, subsequently, a highly resolved, conformer-specific IR spectrum in the 6 mum fingerprint region, using a cold ion trap in combination with table-top lasers. The study has revealed at least three conformational states of GS populated under our experimental conditions, with the major one showing evidence of a symmetric three-dimensional structure similar to that in the condensed phase. The derived qualitative constraints, along with the measured vibrational frequencies, serve as a benchmark for computations of peptide structure.