Article pubs.acs.org/JPCA
IRMPD Spectroscopy: Evidence of Hydrogen Bonding in the Gas Phase Conformations of Lasso Peptides and their Branched-Cyclic Topoisomers Kevin Jeanne Dit Fouque,† Hélène Lavanant,*,† Séverine Zirah,‡ Vincent Steinmetz,§ Sylvie Rebuffat,‡ Philippe Maître,*,§ and Carlos Afonso† †
Normandie Univ, COBRA, UMR 6014, FR 3038; Univ Rouen; INSA Rouen; CNRS, 1 Rue Tesnière, 76821 Mont-Saint-Aignan Cedex, France ‡ Muséum national d’Histoire naturelle, Sorbonne Universités, Centre national de la Recherche scientifique, Laboratoire Molécules de Communication et Adaptation des Microorganismes, UMR 7245 CNRS-MNHN, CP 54, 57 rue Cuvier, 75005 Paris, France § Laboratoire de Chimie Physique, Université Paris Sud, UMR 8000 CNRS, Faculté des Sciences, Bât. 349, 91405 Orsay Cedex, France S Supporting Information *
ABSTRACT: 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.
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INTRODUCTION Lasso peptides constitute a fascinating class of bioactive natural products synthesized by bacteria, which present a mechanically interlocked structure.1 These peptides are stabilized by steric constraints leading to a stable and compact rotaxane type structure. The highly compact lasso topology offers lasso peptides an extraordinary stability toward denaturing conditions and protease degradation.2,3 Because of their high stability and the associated biological activities as antimicrobials, enzyme inhibitors or receptor antagonists,1,4 lasso peptides represent a valuable scaffold for drug design.5,6 Microcin J25 (MccJ25) and capistruin (Figure 1), produced by Escherichia coli AY25 and Burkholderia thailandensis E264, respectively, are two different and representative lasso peptides, that target RNA polymerase.7,8 The structural feature of these lasso peptides consists of an N-terminal macrolactam ring through which the C-terminal tail is threaded and sterically trapped by bulky amino acids.2,9 On the basis of NMR studies, it has been proposed that the lasso structure of MccJ25 and capistruin are stabilized by hydrogen bonds, via short antiparallel β-sheets and an ionic hydrogen bond only for MccJ25.2,9 Several lasso peptides have been reported to unthread under denaturing conditions, yielding their unthreaded branched-cyclic forms.10,11 One important goal is therefore to unambiguously © 2016 American Chemical Society
characterize the mechanically interlocked topologies and differentiate them from their unthreaded branched-cyclic topoisomers. Recently, ion mobility coupled to mass spectrometry (IM− MS) was found very discriminant for lasso peptides and their branched-cyclic topoisomers.12,13 It has been shown that for highly charged [M + 4H]4+ ions of the branched-cyclic peptides only, the CCS values significantly increase, suggesting an unfolding of its C-terminal tail in the gas phase due to Coulombic repulsions (Figure S1). Infrared multiple photon dissociation (IRMPD) spectroscopy is an alternative and complementary approach to IM−MS for providing structural information on mass-selected ions.14 Infrared free electron lasers (IR FEL) and optical parametric oscillator/amplifier (OPO/A) benchtop lasers provide access to a wide frequency range, allowing to record vibrational spectra in the mid-infrared and in the X−H (X = C, N, O) stretching regions, respectively. This so-called action spectroscopy has been particularly successful for distinguishing isomers15,16 and unravelling the hydrogen bonding association to peptide structuration.17,18 Received: May 4, 2016 Revised: May 12, 2016 Published: May 12, 2016 3810
DOI: 10.1021/acs.jpca.6b04496 J. Phys. Chem. A 2016, 120, 3810−3816
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with 4% formic acid to ensure complete protonation. Sulfolane was added up to 0.5% volume. IRMPD Spectroscopy. Mass spectrometry and infrared action spectroscopy experiments were performed employing a 7 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer (Apex Qe, Bruker) coupled with tunable infrared lasers. A detailed layout of this experimental apparatus is described elsewhere.20,21 Mass-selected ions were accumulated in an argon pressurized (∼10−3 mbar) linear hexapole ion trap, thus ensuring thermalization through multiple collisions with argon. Ions were then pulse extracted and stored in the ICR cell where they were irradiated with infrared light. Infrared action spectroscopy was carried out by monitoring the intensities of precursor (Iprecursor) and resulting fragment ions (Ifragment) as a function of the laser wavenumber. The infrared action spectra presented here were obtained by plotting the photodissociation efficiencies, defined as ln (1 + ΣIfragment/Iprecursor), as a function of the laser wavenumber. The most abundant fragment observed upon infrared activation of mass-selected [M + 3H]3+ and [M + 4H]4+ ions of peptides was the loss of a water molecule from the C-terminus (Figures S2 and S3). Other fragment channels which led to very low ion intensities and did not significantly affect the photodissociation efficiencies were not used in the calculations. The ionization source parameters are detailed in Supporting Information. Infrared action spectra of multiply protonated molecules were recorded in the 2800−3700 cm−1 spectral range using an optical parametric oscillator/amplifier (OPO/A from LaserVision, Bellevue, WA) benchtop laser.21,22 Two different OPA settings were used in order to optimize the laser output energy in the 2800−3400 cm−1 and 3400−3700 cm−1 spectral regions. The irradiation time was 1 s. In order to enhance the infrared induced fragmentation efficiency, an auxiliary broadband CO2 laser (BFI Optilas, Evry, France) was used. As described elsewhere,23 a train of CO2 pulses at 25 Hz was generated and synchronized with the 25 Hz tunable infrared laser pulses with a delay of few microseconds. The CO2 laser pulse length was optimized so as to minimize the fragmentation upon CO2-only excitation. The CO2 pulse length was 500 μs and 1 ms for capistruin and MccJ25, respectively, in the 3400−3700 cm−1 spectral range. In the 2800−3400 cm−1 spectral range, the CO2 pulse length was 1.5 and 4 ms for the two lasso and branchedcyclic forms, respectively. Infrared spectroscopy in the 1400− 1800 cm−1 spectral range was performed using the infrared free electron laser (IR FEL, from CLIO, Orsay, France).24 The irradiation time was set to 250 and 500 ms for [M + 3H]3+ and [M + 4H]4+ respectively, to record vibrational spectra in the mid-infrared. These time values were used in order to minimize saturation effects, which can easily occur using the highly intense IR FEL.
Figure 1. Sequences and 3D representations of the structure of a) MccJ25 and b) capistruin. Schemes show the macrolactam rings in green, the C-terminal parts in blue and the plugs in magenta. Relevant functional groups for the present infrared spectroscopic investigation are highlighted in the peptide sequences (yellow, alcohol OH; orange, carboxylic acid OH; green, amide NH2; blue, peptide bond NH; red, peptide bond CO, only made visible here for the isopeptidic bond forming the macrolactam ring).
In this work, we investigated MccJ25, capistruin (Figure 1), and their branched-cyclic topoisomers using IRMPD action spectroscopy. The two spectral ranges provided by the IR FEL and OPO/A benchtop lasers were selected in order to derive spectroscopic information on the hydrogen bonding changes in [M + 3H]3+ and [M + 4H]4+ ions for each type of topoisomer and thus tackle the question of how the hydrogen bond network participates in the stabilization of the mechanically interlocked structures. In the following discussion, a special emphasis will be placed on the consistency of the present spectroscopic results with the previous observations using ion mobility spectrometry.
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EXPERIMENTAL METHODS Peptides and Sample Preparation. MccJ25 was produced from a culture of E. coli MC4100 harboring the plasmid pTUC202,19 cultivated for 16 h in M63 medium. Capistruin was produced from a culture of the naturally producing strain B. thailandensis E264,2 incubated for 24 h at 42 °C in M20 medium containing gentamycin (8 μg/mL). The purification of lasso peptides is detailed in the Supporting Information. The synthetic branched-cyclic peptides, corresponding to the sequences of MccJ25 and capistruin but without threading of the C-terminal tail, were obtained from Genepep (St Jean de Védas, France). Sulfolane was purchased from Sigma-Aldrich (Saint Quentin-Fallavier, France). The 10 μM solutions were prepared in water/acetonitrile 50/50 (v/v)
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RESULTS AND DISCUSSION X−H (X = N, O) groups in lasso or branched-cyclic topoisomers can act as hydrogen bond donors, which results in spectral shifts. The spectral shifts of stretching bands of the amide NH2, amide NH, carboxylic OH and alcohol OH groups can be monitored in the 2800−3700 cm−1 spectral range using the OPO/OPA laser. Two types of hydrogen bonds must be distinguished, however, depending on the charge of the X−H (X = N, O) hydrogen bond donor group. When this group is positively charged, resulting from a protonation of an X lone pair, ionic hydrogen bonds are formed. In view of the charge states of the peptides considered here, it is likely that the most 3811
DOI: 10.1021/acs.jpca.6b04496 J. Phys. Chem. A 2016, 120, 3810−3816
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The Journal of Physical Chemistry A basic residues were protonated and involved in ionic hydrogen bonds. In the case of MccJ25 and its branched-cyclic form, the His5 side chain would most likely be protonated. For capistruin and its branched-cyclic topoisomer, the two protonated Arg11 and Arg15 are also likely to be involved in strong hydrogen bonds. These ionic hydrogen bonds are stronger than neutral hydrogen bonds, thus leading to stronger red-shifts as shown by extensive studies on proton bound dimers resulting from ionic hydrogen bonding patterns.25 It has been shown in particular that large spectral shifts can be observed, and that the resulting X-H stretching bands can be found between 1000 and 3500 cm−1. Considering the wavelength ranges accessible by the two lasers used here, the 2000−2500 cm−1 region could not be probed with sufficient infrared power to induce an IRMPD process. In addition, the spectral shift is accompanied by a broadening, making the bands difficult to observe under our experimental conditions. When a neutral hydrogen bonding between amide NH and CO is at play, spectral shifts in the 1400−1800 cm−1 spectral range explored using the IR FEL should occur concomitantly with spectral shifts observed in the 2800−3700 cm−1 spectral range. More specifically, a blue shift of the NH bending should be associated with a red-shift of carbonyl CO stretching bands. IRMPD Spectroscopy of the Branched-cyclic Form of MccJ25. Infrared action spectra of the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic form of MccJ25 are shown in parts a and b of Figure 2, respectively. Very well-resolved bands were observed, especially in the high energy part of the infrared spectrum, which was not anticipated considering the large size of the peptide containing twenty-one residues. A tentative assignment of the observed infrared bands of the branchedcyclic form of MccJ25 is proposed in Table S1. In the high energy range, the bands observed at ∼3640 cm−1 (highlighted in yellow in Figure 2) were assigned to free alcohol OH stretches. This band can arise from the side chain of serine (S18), threonine (T15), and tyrosine (Y9 and Y20) residues.26,27 The bands observed at ∼3570 cm−1 (highlighted in orange) are characteristic of free carboxylic OH stretches.27,28 The bands observed near ∼3475 cm−1 (highlighted in light blue) are typical signatures of free amide NH stretches of the peptide bonds.29,30 The full width at halfmaximum (fwhm) of these three bands in the high energy range was on the order of ∼15 cm−1, i.e., on the order of magnitude of an IRMPD band associated with a single infrared active mode.31,32 Considering the large size of the peptide, and thus the number of infrared active amide NH groups, the narrow character of these bands is particularly interesting, and suggests that most of the amide NH groups are nearly equivalent. One would have expected a broader profile resulting from several unresolved features, each one being characteristic of amide NH groups in different environments. A clear evidence for changes in the hydrogen bonding pattern between the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic form of MccJ25 could be observed in the 3200−3500 cm−1 spectral range (highlighted in purple in Figure 2, parts a and b). While no band was observed in the case of [M + 4H]4+, a broad band centered at ∼3340 cm−1 was observed for the [M + 3H]3+ ions. This broad band could be assigned to red-shifted amide NH stretches, generally observed between 3300 and 3450 cm−1.18,30,33 This assignment was further supported by the differences in the infrared spectra of
Figure 2. Infrared spectra of (a) [M + 3H]3+ and (b) [M + 4H]4+ ions of the branched-cyclic form of MccJ25 and of (c) [M + 3H]3+ and (d) [M + 4H]4+ ions of MccJ25. The vibrational bands are assigned using a color coding as follows: blue (amide NH bend), red (carbonyl CO stretch), purple (H-bonded amide NH stretch), light blue (free amide NH stretch), orange (free carboxylic OH stretch), and yellow (free alcohol OH stretch). In the 2800−3700 cm−1 spectral range, the gray and black infrared spectra were obtained with a laser power optimized at 3100 and 3660 cm−1, respectively.
[M + 3H]3+ and [M + 4H]4+ ions in the 1400−1800 cm−1 spectral region. The maximum of the NH bending band34 (highlighted in blue in Figure 2, parts a and b) was blue-shifted in [M + 3H]3+ ions as compared to [M + 4H]4+ ions for which the corresponding band is typical of free amide NH bend of the peptide bonds.35,36 Also consistent with a neutral NH···OC hydrogen bonding in [M + 3H]3+ ions was the shape of the CO stretch17,36,37 (highlighted in red). As compared to the [M + 4H]4+ infrared spectrum, an additional red-shifted component was observed in the case of [M + 3H]3+.34,38 These differences in the infrared spectra of the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic peptide indicated that the gas phase conformation of the [M + 3H]3+ ions involved additional hydrogen bond interactions compared to the highly charged [M + 4H]4+ ions, for which no neutral hydrogen bonds were evidenced. It should be kept in mind, however, that ionic hydrogen bond interactions involving in particular the His5 residue may also be at play, and that the corresponding spectral shifts cannot be probed using our experimental setup. The differences in the infrared spectra of the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic topoisomers are consistent with the ion mobility spectrometry observations.13 Indeed, the CCS values of the branched-cyclic 3812
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red-shifted NH stretches in the 3300−3400 cm−1 spectral range.41 The similar infrared spectra of the [M + 3H]3+ and [M + 4H]4+ ions of the lasso peptides suggest that the gas phase conformation of the hydrogen bond interactions are not strongly affected by the charge state. Together with the similar CCS values (Table S1) of the [M + 3H]3+ and [M + 4H]4+ ions of the lasso peptides,13 these observations suggest that the [M + 3H]3+ and [M + 4H]4+ ions of the lasso peptides have similar conformations. For all charge states, lasso peptides remain compact due to the mechanical hindrances imposed by the bulky residues in addition to neutral hydrogen bonding interactions and an ionic hydrogen bonding that strengthen the lasso structure. IRMPD Spectroscopy of the Branched-Cyclic Form of Capistruin. Infrared action spectra of the [M + 3H]3+ and [M + 4H]4+ charged ions of the branched-cyclic form of capistruin are presented in Figure 3a and 3b, respectively. A tentative assignment of the observed infrared bands is proposed in Table S2. As compared to the branched-cyclic form of MccJ25, the 2800−3700 cm−1 infrared region is more difficult to assign because capistruin has an additional type of functional group:
topoisomer (Table S1) are consistent with a more compact structure for the [M + 3H]3+ ions than for the [M + 4H]4+ ions. As compared to the [M + 4H]4+ ions, the folding of the Cterminal tail in the gas phase conformation of the [M + 3H]3+ ions would involve additional hydrogen bonding, which would be consistent with the spectral shifts assigned to the abovediscussed NH···OC hydrogen bonding. IRMPD Spectroscopy of MccJ25. Infrared action spectra of the [M + 3H]3+ and [M + 4H]4+ ions of MccJ25 lasso peptide are presented in Figure 2, parts c and d, respectively. Hydrogen bonding between X−H (X = N, O) and carbonyl CO groups could also be expected in this case. Indeed, the combined NMR and molecular mechanic study9 suggested that the structure of the MccJ25 is characterized by a short antiparallel β-sheet composed of residues Phe10-Val11 and Thr15-Pro16 that involve neutral hydrogen bonds between amide NH and CO groups. Furthermore, the same study found the His5 residue to be protonated and very close to the Cterminal carboxylate suggesting that an ionic hydrogen bond may stabilize the structure. As for the branched-cyclic form of MccJ25, the lasso topology also displayed well resolved bands, especially in the high energy part of the infrared spectrum. A tentative assignment of the observed infrared bands is proposed in Table S1. As for the branched-cyclic form, the infrared spectra of [M + 3H]3+ and [M + 4H]4+ ions of MccJ25 in the 3450−3700 cm−1 region were found very similar, both in terms of band positions and profiles. In fact, free alcohol OH stretch, free carboxylic acid OH stretch and free amide NH stretch bands were observed for each charge state and are highlighted in yellow, orange and light blue, respectively, in Figure 2c and 2d. Considering the number of infrared active XH groups, the narrow bandwidth (∼15 cm−1) is particularly interesting, and suggests, as for its branched-cyclic form, that several amide NH groups are nearly equivalent and “free”. Evidence for hydrogen bonding in both [M + 3H]3+ and [M + 4H]4+ ions could be observed in the 3200−3500 cm−1 spectral range (highlighted in purple in Figure 2, parts c and d). In the case of the [M + 3H]3+ ions, an unresolved band with two maxima at 3340 and 3420 cm−1 could be observed. A similar feature could be observed for the quadruply charged ions, although the signal-to-noise was low due to lower ion abundance. This hydrogen bonding hypothesis was further supported by the 1400−1800 cm−1 spectral region, which showed comparable profiles for the two charge states. If the amide NH bending mode (highlighted in blue in Figure 2, parts c and d) is considered, in particular, the bandwidth and the position of the band maximum are the same for both the triply and quadruply charged MccJ25. This suggests that, contrary to the case of the above-discussed branched-cyclic topoisomer, the hydrogen bonding network involving the amide NH hydrogen bond donor does not strongly change, from the triply to the quadruply charged peptide. Similar conclusions could be drawn from the comparison of the infrared spectra in the CO stretching region (highlighted in red). It should be stressed that due to the complex processes associated with the multiple photon absorption leading to fragmentation of IRMPD, relative fragmentation intensities may poorly reflect relative absorption intensities as predicted by theory.39,40 Nevertheless, these spectral features seem to be consistent with the proposed structure of the lasso peptide exhibiting two antiparallel β-sheet domains. Indeed, this secondary structure is characterized in this case by two amide I bands at ∼1615 and 1675 cm−1, and
Figure 3. Infrared spectra of (a) [M + 3H]3+ and (b) [M + 4H]4+ ions of the branched-cyclic form of capistruin and of (c) [M + 3H]3+ and (d) [M + 4H]4+ ions of capistruin. The vibrational bands are assigned using a color coding as follows: blue (amide NH bend), red (carbonyl CO stretch), purple (H-bonded amide NH stretch), green (free amide NH2 symmetric and asymmetric stretch), light blue (free amide NH stretch), pink (H-bonded OH stretch), and yellow (free alcohol OH stretch). In the 2800−3700 cm−1 spectral range, the gray and black infrared spectra were obtained with a laser power optimized at 3100 and 3660 cm−1, respectively. 3813
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spectrum, no IRMPD signal was observed in the region of the free alcohol OH stretch band near ∼3650 cm−1 in the cases of the two charge states. This lack of free alcohol OH band suggests that serine (S14) and threonine (T2 and T7) residues may be involved in hydrogen bonding interactions. More importantly, evidence for hydrogen bonding in both [M + 3H]3+ and [M + 4H]4+ ions could be found with a broad band at ∼3360 cm−1 (highlighted in purple in Figure 3, parts c and d). As for MccJ25, this broad band could be interpreted as a signature of a neutral NH..OC hydrogen bond, in line with the antiparallel β-sheet domain proposed by Knappe and coworkers.2 In fact, they have shown that the structure of capistruin is characterized by a short antiparallel β-sheet composed of residues Thr7-Pro8 and Ile13-Ser14 involving neutral hydrogen bonds between NH and CO groups. The hypothesis that the hydrogen bonding interaction is not strongly affected by the charge state of capistruin was further supported by the similarity of the infrared spectra of [M + 3H]3+ and [M + 4H]4+ ions in the 1400−1800 cm−1 spectral region. Both the amide NH bending band (highlighted in blue in Figure 3, parts c and d) and the CO stretching band (highlighted in red) have the same profile for the two charge states. As for MccJ25, it can thus be concluded that the [M + 3H]3+ and [M + 4H]4+ ions of capistruin both present similar neutral hydrogen bond interactions. These similarities were consistent with the ion mobility-mass spectrometry observations. In fact, the similar CCS values (Table S2) of the lasso topology are consistent with similar gas phase conformations for [M + 3H]3+ and [M + 4H]4+ ions. For all charge states, capistruin remains compact due to the bulky residues and neutral hydrogen bonds between NH and CO groups and OH hydrogen bonds interactions that strengthened the lasso structure.
the NH2 carried by the arginine (R11 and R15), asparagine (N19), and glutamine (Q6) residues. The unresolved band with maxima at ∼3440 and ∼3470 cm−1 could be assigned to NH stretches. The latter (highlighted in light blue in Figure 3) is a typical signature of free amide NH stretches of peptide bonds. The former (highlighted in green) at ∼3440 cm−1 could be tentatively assigned to free amide NH2 symmetric stretches.30 In the higher energy part of the spectrum, free amide NH2 asymmetric stretches and free OH stretches are expected near 3550 and 3650 cm−1, respectively. Contrary to MccJ25, no signal corresponding to free alcohol OH stretches (highlighted in yellow in Figure 3a) near ∼3640 cm−1 was observed for the [M + 3H]3+ ions of the branched-cyclic form of capistruin. These OH groups are characteristic of the side chain of serine (S14) and threonine (T2 and T7) residues. The fact that no free OH could be detected for the [M + 3H]3+ ions of the branched-cyclic form of capistruin suggests that OH groups were likely involved in hydrogen bond interactions. As a result, the maximum near ∼3550 cm−1 is tentatively assigned to free amide NH2 asymmetric stretches, and the shoulder on its red-side could be assigned to slightly red-shifted alcohol OH stretches (highlighted in pink in Figure 3), generally observed between 3200 and 3550 cm−1.32,42 As for the branched-cyclic form of MccJ25, there was converging evidence pointing at a change in the hydrogen bonding pattern between the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic form of capistruin. While a low IRMPD signal was observed in the 3200−3500 cm−1 (highlighted in purple in Figure 3a and 3b) in the case of [M + 4H]4+, a broad band centered at ∼3350 cm−1 was observed for the [M + 3H]3+ ions. The emergence of this broad band when going from the quadruply to the triply charged ion was correlated with changes in the 1400−1800 cm−1 region which are consistent with neutral NH···OC hydrogen bonding interactions. First, the amide NH bend (highlighted in blue in Figure 3a) was blueshifted, and second,the CO stretching band (highlighted in red in Figure 3a) had a red-shifted component in the case of [M + 3H]3+ as compared to [M + 4H]4+ for which the corresponding band at ∼1655 cm−1 is typical of a free carbonyl CO stretch of peptide bonds. In addition, the emergence of a free alcohol OH stretching band at ∼3650 cm−1 (highlighted in yellow in Figure 3b) in the case of [M + 4H]4+ ions, also suggested that one or more alcohol OH group were freed, as compared to the [M + 3H]3+ ions. These differences in the infrared spectra of the [M + 3H]3+ and [M + 4H]4+ ions of the branched-cyclic topoisomer indicated that the gas phase conformation of the [M + 3H]3+ ions involved additional hydrogen bond interactions than its highly charged [M + 4H]4+ ions. These differences are consistent with the ion mobility spectrometry observations.13 Indeed, the CCS values of the branched-cyclic topoisomer (Table S2) show a more compact structure for the [M + 3H]3+ ions than for the [M + 4H]4+ ions. As compared to the [M + 4H]4+ ions, the folding of the C-terminal tail in the gas phase conformation of the [M + 3H]3+ ions would involve additional hydrogen bonding, which would be consistent with the spectral shifts assigned to the above-discussed NH···OC hydrogen bonding. IRMPD Spectroscopy of Capistruin. As for MccJ25, infrared spectra of [M + 3H]3+ and [M + 4H]4+ ions of capistruin were found quite similar (Figure 3, parts c and d), and a tentative assignment of the observed infrared bands is proposed in Table S2. In the high energy part of the IR
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CONCLUSION The present spectroscopic data are fully consistent with our previous ion mobility data. They provide strong evidence for a correlation between smaller/larger collision cross sections and hydrogen-bond making/breaking. When going from a triply to a quadruply protonated peptide, the IM−MS data suggest an unfolding of the branched-cyclic topoisomers only, which is consistent with the evolution of the infrared spectrum of the branched-cyclic peptide in both the amide I−II and NH stretching regions from the triply and quadruply protonated state. The previous IM−MS and the present spectroscopic data are also consistent with the well-known high stability of lasso peptides toward denaturing conditions. Upon protonation of the [M + 3H]3+, the small increase of the collision cross section observed with IM−MS suggests very little unfolding. Interestingly, this stability of the compact structure of the lasso peptide is corroborated by the similarity of the infrared spectra in both the amide I−II and NH stretching regions observed for the two lasso peptides (MccJ25 and capistruin) for the two [M + 3H]3+ and [M + 4H]4+ protonation states. IM−MS and IRMPD spectroscopy are two orthogonal methods for deriving structural information on gas phase molecular ions such as multiprotonated peptides under tandem mass spectrometry conditions. Collision cross section obtained through IM−MS provides an information on the global shape of the molecules, and thus on the folding/unfolding in the case of peptides. IRMPD spectroscopy provides information on the presence of hydrogen bonding. These two complementary types of data which showed a clear correlation in the present 3814
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The Journal of Physical Chemistry A
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case, could be used in conjunction with molecular modeling to achieve new insights on intrinsic gas phase molecular structure. Such a potential has motivated the recent development of instrument integrating both ion mobility separation and optical spectroscopy, either in the infrared14,43 or UV−visible44−47 regions.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpca.6b04496. Experimental methods including purification of lasso peptides and ESI source parameters, CCS ranges of MccJ25, capistruin, and their branched cyclic topoisomers (Figure S1), mass spectra and IRMPD mass spectra of MccJ25 and capistruin (Figures S2 and S3), and tentative assignments of experimental vibrational frequencies of MccJ25, capistruin and their branchedcyclic topoisomers (Table S1 and S2) (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*(H.L.) E-mail,
[email protected]. Telephone, +33 2 35 52 29 32. *(P.M.) E-mail,
[email protected]. Telephone, +33 1 69 15 32 50. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by the European Regional Development Fund (ERDF) No. 31708, the Région Haute Normandie (Crunch Network, No. 20-13) and the Labex SynOrg (ANR11-LABX-0029). Financial support from the national FT-ICR network (FR 3624 CNRS) for conducting the research is also gratefully acknowledged.
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REFERENCES
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