Investigation of Secondary Structure Elements by IR/UV Double Resonance Spectroscopy:  Analysis of an Isolated β-Sheet Model System

Determination of intrinsic three-dimensional structures of biomolecules challenges spectroscopy to provide detailed, conformer-selective fingerprints of these large species in the gas-phase. Infrared-ultraviolet (IR-UV) double resonance (DR) spectroscopy has proven to be a powerful tool for measurement of conformation-selective vibrational spectra of polyatomic molecules, [1][2][3][4][5][6][7] ions, [8][9][10][11][12] and their clusters. [9,[13][14][15][16] The technique is based on the change of the vibrational population of the molecule by an IR laser pulse with subsequent detection of this change by a UV laser pulse. DR spectroscopy, when combined with UV photofragmentation, electrospray ionization, and cryogenic cooling, has allowed the detection of IR spectra of large protonated species in the gas phase. [8,9,11,12,17] The opened questions remain, however, how truly these spectra reflect absorption for large species and what are the limitations of this technique. Here we analyze the mechanism of IR-UV DR photofragmentation spectroscopy in its application to large, protonated molecules cooled to cryogenic temperatures. We demonstrate the use of depletion spectroscopy for the unambiguous assignment of vibronic transitions to different conformers of a protonated decapeptide and for the measurement of absolute absorption cross-sections of vibrational transitions in the same species. Finally we extend DR spectroscopy to an intact, protonated protein.Our experimental setup has been described in detail elsewhere. [18,19] Briefly, we produce protonated gas-phase molecules directly from solution using electrospray ionization. The ions of interest are selected by a quadrupole massfilter, guided to a cold (6 K), 22-pole ion trap, where they are cooled by collisions with He. The cold ions are then interrogated by an IR optical parametric oscillator (OPO) and a UV laser. Charged UV-induced photofragments are detected in a quadrupole mass spectrometer. Figure 1 (blue trace) shows an electronic spectrum of the doubly protonated decapeptide gramicidin S, [GS + 2H] 2+ , with several peaks that have been previously assigned to three different conformers by IR-UV depletion spectroscopy. [17,20] In those experiments we fixed the wavenumber of the UV photodissociation laser on different peaks of the blue trace in Figure 1, each time recording depletion of the ion signal as a function of wavenumber of the preceding IR pulse. Difference in the IR spectra measured for some UV peaks (labeled A, B, and C in Figure 1) allowed their conformational assignment. To assign the numerous remaining peaks we reverse the experiment and scan the UV laser wavenumber whereas the wavenumber of the IR OPO is fixed to excite only the two most abundant conformers, labeled A and B. Figure 1 (pink trace) illustrates the changes induced by this vibrational pre-excitation. All sharp peaks, including those assigned to the conformers A and B, are almost completely suppressed, indicating that the IR pulse significantly depletes the vibrational ground states ...

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Exploring the Mechanism of IR–UV Double‐Resonance for Quantitative Spectroscopy of Protonated Polypeptides and Proteins

2013

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“…Even though one may argue that biomolecules are most naturally studied in an aqueous environment, it is meaningful to start from isolated species to which one may later add an increasing number of water molecules to compare their physical data with quantum chemical models . Additionally, gas phase studies of biomolecules enable the elucidation of intrinsic folding preferences without interference of solvents or other molecules …”

Section: Introductionmentioning

confidence: 99%

“…[42,43] Additionally, gas phase studies of biomolecules enable the elucidation of intrinsic folding preferences without interference of solvents or other molecules. [44][45][46][47] While some biomolecules or biomolecular moietiessuch as nucleobases and some vitaminscan be readily sublimated or evaporated [48] oligopeptides, proteins and oligonucleotides will rather fragment than fly when heated. To suppress fragmentation, one may reduce the heating time or add collisional cooling, once the molecules are airborne.…”

Section: Introductionmentioning

confidence: 99%

Tailoring the volatility and stability of oligopeptides

et al. 2017

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Amino acids are essential building blocks of life, and fluorinated derivatives have gained interest in chemistry and medicine. Modern mass spectrometry has enabled the study of oligo‐ and polypeptides as isolated entities in the gas phase, but predominantly as singly or even multiply charged species. While laser desorption of neutral peptides into adiabatically expanding supersonic noble gas jets is possible, UV–VIS spectroscopy, electric or magnetic deflectometry as well as quantum interferometry would profit from the possibility to prepare thermally slow molecular beams. This has typically been precluded by the fragility of the peptide bond and the fact that a peptide would rather ‘fry’, i.e. denature and fragment than ‘fly’. Here, we explore how tailored perfluoroalkyl functionalization can reduce the intermolecular binding and thus increase the volatility of peptides and compare it to previously explored methylation, acylation and amidation of peptides. We show that this strategy is essential and enables the formation of thermal beams of intact neutral tripeptides, whereas only fragments were observed for an extensively fluoroalkyl‐decorated nonapeptide. © 2017 The Authors. Journal of Mass Spectrometry Published by John Wiley & Sons Ltd.

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“…One of these interactions occurring between amino and carbonyl groups of amide functionalities within the backbone are hydrogen bonds, whose characteristic patterns are responsible for typical secondary structures (e.g. [18,[23][24][25][26][27][28][29][30][31]. Beyond these hydrogen bonds, additional interactions including the side chains are of relevance, such as a covalent disulphide bond between two cysteine residues which has a strong impact on tertiary structures.…”

Section: Introductionmentioning

confidence: 99%

Isolated β‐Turn Model Systems Investigated by Combined IR/UV Spectroscopy

et al. 2012

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The functionality of bioactive molecules sensitively depends on their structure. For the investigation of intrinsic structural properties, molecular beam experiments combined with laser spectroscopy have proven to be a suitable tool. Herein we present an analysis of the two isolated tripeptide model systems Ac-Phe-Tyr(Me)-NHMe and Boc-Phe-Tyr(Me)-NHMe. For this purpose, mass-selective combined IR/UV spectroscopy is applied to both substances in a molecular beam experiment. The comparison of the experimental data with DFT calculations, including different functionals as well as dispersion corrections, allows an assignment of both tripeptide models to β-turns formed independently from the protection groups and supported by the interaction of the two aromatic chromophores.

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Investigation of Secondary Structure Elements by IR/UV Double Resonance Spectroscopy: Analysis of an Isolated β-Sheet Model System

Cited by 62 publications

References 46 publications

Exploring the Mechanism of IR–UV Double‐Resonance for Quantitative Spectroscopy of Protonated Polypeptides and Proteins

Exploring the Mechanism of IR–UV Double‐Resonance for Quantitative Spectroscopy of Protonated Polypeptides and Proteins

Tailoring the volatility and stability of oligopeptides

Isolated β‐Turn Model Systems Investigated by Combined IR/UV Spectroscopy

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