Ana Isabel
González Flórez
,
Doo-Sik
Ahn
,
Sandy
Gewinner
,
Wieland
Schöllkopf
and
Gert
von Helden
*
Fritz-Haber-Institut der Max-Planck-Gesellschaft, Faradayweg 4-6, 14195 Berlin, Germany. E-mail: helden@fhi-berlin.mpg.de
First published on 28th July 2015
Ultracold IR spectra of the protonated five amino acid peptide leu-enkephalin (Tyr-Gly-Gly-Phe-Leu) embedded in superfluid helium droplets have been recorded using a free-electron laser as radiation source. The results show resolved spectra, which are in good agreement with theoretical calculations, as well as with the available gas-phase data indicating that the helium environment does not induce a significant matrix-shift. In addition, the effect of the interaction between the charge and the peptide backbone has been further investigated by complexing protonated leu-enkephalin with one 18-crown-6 molecule. Good agreement between the experimental and theoretical results allow for an assignment of a preferred molecular structure.
To investigate peptides or other biological molecules free of solvation, they can, for example, be studied as neutral molecules in molecular beams using various laser spectroscopic methods.1–4 While those experiments prove to be highly successful and can yield detailed information, they get quite difficult for larger species.
In a more general approach, charged species can be investigated by combining mass spectrometry with optical spectroscopy. In particular, infrared (IR) spectroscopy on mass-to-charge selected species has become a powerful tool to elucidate the structure of small, as well as large peptide and protein ions.5 In a commonly used implementation, the gas-phase ion of interest is irradiated by an intense IR pulse. When the light is resonant with an allowed infrared transition, the absorption of multiple photons can take place, leading to fragmentation. Monitoring the fragmentation as a function of IR wavelength gives an IR spectrum. A drawback of this so called IR multiple photon dissociation (IRMPD) scheme is that peaks in the spectrum get broadened and shifted, and that their relative intensities depend on the mode anharmonicities and on the presence of neighbouring absorptions.6 To circumvent those limitations, techniques to investigate biomolecular ions in cooled ion traps have been developed that require only the absorption of one or a few photons. When performing IR/UV double resonance experiments in a cold trap, changes in UV fragmentation yield upon absorption of IR photons are recorded.7–10 However, the required presence of a UV chromophore limits the applicability of these methods. In another approach, weakly bound adducts of small messenger molecules (e.g. rare gases) are formed, which dissociate from the ion after IR absorption.11,12 Here again, measuring the dissociation yield as a function of wavelength gives then an IR spectrum.
An alternative method to cool ions is to embed the species of interest in liquid helium nanodroplets.13 Those droplets are ultracold, and provide with an equilibrium temperature of ≈0.4 K, an efficient heat bath that is superfluid, only weakly interacting with the dopant and optically transparent from the deep-UV to the far-IR. Because of those properties, liquid helium droplets are considered an ideal matrix for spectroscopic experiments and have been used to investigate a variety of small molecules and molecular ions.14 In most experiments, dopant molecules are thermally evaporated and picked up by the droplets. However, with the exception of very small species, biological molecules cannot be thermally evaporated and those established methods can not be employed. We devised a scheme in which ions that are brought into the gas phase by electrospray ionization and mass-to-charge selected by a quadrupole mass spectrometer are picked up from an ion trap by helium droplets.15 It has been shown that this method can be used to pick up species as large as an entire protein.15 This method can be used to perform spectroscopy and the UV spectrum of mass-to-charge selected [Hemin + H]+ has been reported.16
Here, we apply helium droplet isolation IR spectroscopy to the mass-to-charge selected protonated pentapeptide leu-enkephalin (Tyr-Gly-Gly-Phe-Leu, Leu-Enk) as well as to its complex with one 18-crown-6 ether molecule. Leu-Enk is an endogenous opioid neurotransmitter peptide that can be found in many living organisms. As it is small enough to allow its detailed investigation, yet large enough to be a realistic model peptide, Leu-Enk has been studied using many gas-phase techniques, including mass spectrometry17,18 and optical spectroscopy.9,10,19 Using IR/UV double resonance spectroscopy in a cold ion trap, a high resolution IR spectrum has been recorded, allowing for structure assignment10 and it is found that the structure is dominated by the intramolecular solvation of the charged N-terminus by carbonyl groups. Here, we focus on protonated Leu-Enk as a test case for IR spectroscopy in helium droplets and crown-ether complexed Leu-Enk to investigate the role of internal charge solvation in the molecule.
Helium droplets are produced using a cryogenic Even–Lavie pulsed valve, cooled by a closed cycle cryostat (Sumimoto RDK 408D2). The valve is operated with a repetition rate of 10 Hz and a stagnation pressure of 70 bar. Due to velocity spread, the broadening of each pulse of helium droplets within the laser-interaction region of our instrument is measured to be ≈80 μs. The average size of the generated droplets can be varied by adjusting the nozzle temperature, which is set to 25 K in the current experiment. Under these conditions, helium droplets have a typical size of about 20000 He atoms. After passing through a skimmer with a diameter of 4 mm placed 13.5 cm away from the nozzle, the beam of helium droplets traverses the ion trap where ion-pickup can take place. Due to their large kinetic energy given by their mass and the beam velocity (≈500 m s−1), the ion-doped helium droplets can overcome the small longitudinal DC trapping potential. Further downstream, the doped droplets can either be detected directly using a Daly type detector or probed using the Fritz Haber Institute IR-free-electron laser (FHI FEL).20 In the latter case, the IR laser overlaps the doped droplet beam counter propagating in the extraction region of a small linear time-of-flight (TOF) mass spectrometer built in-house. The laser beam is mildly focused and converging and has an estimated diameter of 5 to 8 mm during overlap with the droplet beam.
Table S1 (ESI†) lists the (zero point energy corrected) relative B3LYP-D3 energies of those 24 structures, many of which are low in energy. Since they are obtained by straight energy minimization of the MD structures at the B3LYP-D3 level with no further annealing of the orientations of the side-chains or the puckering of the 18-crown-6 molecule, the structures might not be the global minimum structure within their respective hydrogen bond constraint group. The relative energies should therefore just be regarded as a guide.
A typical time-of-flight (TOF) mass spectrum of the ejected ions after resonant IR excitation of droplets doped with protonated leu-enkephalin [Leu-Enk + H]+ is depicted in Fig. 2a. The signal at m/z ≈ 556 corresponds to [Leu-Enk + H]+, while the peak of small intensity observed at the higher m/z possibly results from a water adduct [Leu-Enk + H2O + H]+. It is observed that fragmentation of [Leu-Enk + H]+ can be induced by very high photon densities. On the other hand, when the photon density is reduced, the signal corresponding to [Leu-Enk + H]+ and its water adduct diminishes and eventually disappears. However, the remainder of the helium solvation is under no circumstances observed. This is in accordance with the ejection of the ions from the droplet and is difficult to rationalize with a picture in which dopant ions are observed due to the complete evaporation of the helium atoms from the droplet. Furthermore, the total absence of helium adducts in the TOF spectra independent of the photon density suggests a very low interaction between the embedded molecule and the surrounding helium environment.
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Fig. 2 (a) TOF mass spectrum of the ejected ions from the helium droplets upon IR photo excitation. (b) Signal of the ejected ion as a function of laser power. |
Fig. 2b shows the signal of the ejected ion as a function of the photon density when excitation occurs at 1530 cm−1 (Amide II) and 1700 cm−1 (Amide I). The laser power dependence is clearly non-linear, implying that the absorption of more than one photon is required in order to eject the ion. A non-linear character of the ejection efficiency of the embedded ion has been previously reported16 when exciting with UV light. In this experiment, it was shown that the absorption of at least two 378 nm photons is required to efficiently eject [Hemin + H]+ from the doped droplet. Fig. 2b further shows that the ejection efficiency depends on the excitation wavelength.
The efficiency of the ion ejection as a function of both, laser power and wavelength, is an important parameter to take into account when recording wavelength spectra of dopant molecules. The output power of the FEL depends strongly on the undulator settings, the precise tuning of the electron beam, the laser cavity, and other parameters. Thus, while scanning over a wide wavelength range, variations in output power will occur which will influence the intensity of the signal. Since the exact laser power dependence of the ejection efficiency is not known, all wavelength spectra shown in the remainder are simply divided by the respective laser power curves that are measured before and after each scan as a first order correction.
He droplets (cm−1) | Cold trap10 (cm−1) | Calc. (cm−1) | Assignment |
---|---|---|---|
1280 | 1285 | Tyr ring deform. | |
1421 | 1422 | Tyr ring deform. | |
1451 | 1467 | ||
1498 | 1502 | Tyr ring deform. | |
1507 | Sym. NH3+ | ||
1523 | 1529 | 1520 | N–H bend |
1600 | Antisym. NH3+ | ||
1619 | Tyr ring deform. | ||
1623 | 1631 | 1643 | Phe C![]() |
1666 | 1675 | 1682 | Gly2 C![]() |
1695 | 1689 | 1693 | Gly1 C![]() |
1702 | 1710 | 1711 | Tyr C![]() |
1722 | 1730 | 1722 | Leu C![]() |
1744 |
The experimental results displayed in the upper panel of Fig. 3b are in good accord with the calculated transitions. In contrast to the spectrum of the undeuterated molecule, two bands are now observed in the amide II region at 1460 cm−1 and 1518 cm−1, respectively. A complete summary of transition frequencies is reported in Tables S2 and S3 (ESI†). Considering the dominant CO stretch character of the amide I band, one would expect essentially no changes in the amide I region of the experimental spectrum upon deuteration. However, in addition to a minor redshift of less than 10 cm−1, a more convoluted band at 1694 cm−1 is observed. In order to understand this observation one has to take into account that the C
O stretches typical of this spectral region are not pure amide I transitions, but C
O's which are coupled with other vibrations, such as C–H and N–H bending modes. The effect of deuteration on the IR spectrum is also small for the band observed in the amide III region, which is only slightly shifted towards lower wavenumbers.
The most dramatic changes upon deuteration can be found in the amide II region, where the single band observed at 1523 cm−1 in the spectrum of [Leu-Enk + H]+ is now split into two well separated transitions; one of them undergoes a significant redshift (−63 cm−1) while the other remains at a very similar wavenumber position (−5 cm−1). The calculations for the undeuterated molecule show that, in addition to the N–H bending modes, aromatic ring deformation modes are active in this region. The N–D modes will therefore shift and give rise to the band at 1460 cm−1 while ring deformation modes will remain. Those exhibit the largest blue-shifts with respect to the calculations (≈+20 cm−1), which is in line with the results obtained for the undeuterated molecule [Leu-Enk + H]+.
The –NH3+ group in [Leu-Enk + CE + H]+ is assumed to only interact with the CE molecule and is therefore not available as a hydrogen bond donor. Likewise, the CE molecule is assumed to exclusively interact with the –NH3+ group. Thus the [Leu-Enk + CE + H]+ molecule has six groups that can act as hydrogen bond donors (Fig. 4a): the Tyr–OH group (labeled OHT), four amide N–H groups (labeled NH1–NH4, from the N- to the C-terminus) and the OH from the C-terminal carboxyl group (labeled OHA). Five CO groups, ranging from the carbonyl group near the N-terminus to the C
O of the C-terminal carboxyl group, can act as hydrogen bond acceptors (labeled O1–O5, see Fig. 4a). While the –OH oxygen can also interact as a hydrogen bond acceptor, those interactions are assumed to be weaker and are not considered. When being in trans conformation, no hydrogen bond between a C
O and N–H group within a peptide bond can be formed. Furthermore, due to steric constraints, an interaction of the OHT with the first carbonyl (O1) is unlikely.
![]() | ||
Fig. 4 Two most favoured calculated structures of [Leu-Enk + CE + H]+ with relative (zero point corrected) energies of 0 kJ mol−1 (a) and 14.8 kJ mol−1 (b). |
Considering the hydrogen bond acceptors and donors described above yields 24 hydrogen bond donor–acceptor permutations for which the corresponding hydrogen bond is constrained to be of a fixed length of 2 Å during the simulation. We compared all 24 simulated IR spectra with our experimental results. The spectrum that gives the best match to the experiment resulted from the simulation in which the hydrogen bond length between the first carbonyl group (O1) and the second NH group (NH2) was constrained. Its structure is shown in Fig. 4b, and its corresponding vibrational spectrum in Fig. 5a. This conformer is +14.8 kJ mol−1 higher in energy than the lowest energy structure, due to a constraint between the second carbonyl group (O2) and the Tyr–OH group (OHT), which is shown in Fig. 4a, with its corresponding spectrum in Fig. 5a.
The backbone of the structure shown in Fig. 4b (+14.8 kJ mol−1) exhibits four internal H-bonds. Common features of this structure and the one in Fig. 4a, are one of the C7 and the C10 rings. However, the OH group of the Tyr residue is no longer hydrogen bonded to the carbonyl of the second Gly residue. Instead, there is an additional C7 ring that connects the amide NH on the first Gly residue with the Tyr carbonyl, as well as a C14 ring between the amide NH of the Tyr residue and the carbonyl of the Leu residue.
The calculated vibrational transitions for both structures are displayed in Fig. 5a, and a list of the transition frequencies with their corresponding intensities can be found in Table 2 and Table S4 (ESI†). In the wavelength range between 1200 cm−1 and 1400 cm−1, both calculations predict several weak transitions, as well as a stronger transition at 1280 cm−1 and 1276 cm−1 for the +14.8 kJ mol−1 and 0 kJ mol−1 structures, respectively. These transitions originate from a mixture of the Tyr ring deformation, C–H, and O–H bending modes. The stronger mode might correspond to the band experimentally observed at 1253 cm−1, although the shift of ≈−30 cm−1 seems rather large. The calculated vibrational transitions of both structures predict a bending mode of the OH group of the Leu residue at 1400 cm−1 and 1430 cm−1 for the +14.8 kJ mol−1 and 0 kJ mol−1 structure, respectively. The difference of ≈30 cm−1 is the result of the OH hydrogen bond between the Tyr–OH group and the carbonyl oxygen of the Gly2 residue in the 0 kJ mol−1 structure. Experimentally, a corresponding small peak that is barely above the noise level is observed at 1402 cm−1. The predicted amide II region of both structures contains closely spaced N–H bending modes and a Tyr-ring deformation mode. These modes are similar in intensities and wavenumbers for both structures. In the amide I region between 1650 cm−1 and 1800 cm−1, both calculations predict five transitions corresponding to the five CO oscillators in the molecule. Their positions and relative intensities differ from each other, reflecting the differences in their hydrogen bonding patterns. Both calculations predict the mode of the Leu C
O group as appearing most to the blue in the spectrum. The H-bond in the +14.8 kJ mol−1 structure causes a red-shift of this mode of −13 cm−1 with respect to the 0 kJ mol−1 structure. Another significant difference between the two calculations stems from the Tyr C
O mode, which for the +14.8 kJ mol−1 structure is shifted towards the red by 30 cm−1, again due to its involvement in a hydrogen bond.
Exp. (cm−1) | Calc. (cm−1) | Intensity (km mol−1) | Assignment |
---|---|---|---|
1253 | 1280 | 134.10 | Tyr ring deform. |
1402 | 1400 | 251.20 | Leu O–H bend |
1505 | 1493 | 117.06 | N–H bend |
1507 | 130.93 | Tyr ring deform. | |
1526 | 1516 | 176.59 | N–H bend |
1522 | 248.26 | N–H bend | |
1528 | 131.66 | Sym. NH3+ umbrella | |
1540 | 148.97 | N–H bend | |
1609 | 60.32 | Antisym. NH3+ umbrella | |
1622 | 76.66 | Tyr ring deform. | |
1678 | 1662 | 120.13 | Phe C![]() |
1691 | 1675 | 112.14 | Gly1 C![]() |
1705 | 1692 | 465.32 | Tyr C![]() |
1725 | 1715 | 520.60 | Gly2 C![]() |
1748 | 1744 | 373.44 | Leu C![]() |
After a comparison of the experimental results with the calculated vibrational transitions, it is evident that the theoretical spectrum of the 14.8 kJ mol−1 structure provides the best agreement with the experimental spectrum of the CE-complexed molecule. The experimentally observed positions and the almost equidistant spacing between the five modes in the amide I range are correctly reproduced by the spectrum of the +14.8 kJ mol−1 structure while the experimental resolution of only four modes would be expected in the spectrum of the 0 kJ mol−1 structure. The amide II region is less informative as both calculations reproduce the experiment. Provided that the weak band at 1402 cm−1 stems from a real transition in the experimental spectrum, the best agreement is given as well by the 14.8 kJ mol−1 structure. The stronger mode calculated in the amide III region is at essentially the same position for both structures and is also ≈30 cm−1 higher in wavenumber than the next corresponding experimental transition.
A comparison between the H-bonding arrangements of the assigned structures of [Leu-Enk + H]+ (available in ref. 10) and [Leu-Enk + CE + H]+ (Fig. 4b) shows that the H-bonds involving as donors the –NH3+ group are no longer present when a CE molecule is added. However, the remaining four H-bonds that are not directly coordinated to the protonated amine site are maintained. Burke et al.10 discussed the importance of charge and charge solvation on the structure and on the CO oscillator vibrational frequencies. According to their analysis, the solvation of the charge allows a network of H-bonds that, in some cases, cooperatively strengthen one another. This is the case for the C
O groups of the Leu and Gly2 residues due to their direct coordination to the –NH3+ group. The H-bond of the Phe C
O, which is coordinated to the Leu OH is also strengthened indirectly via the coordination of the –NH3+ group to the Leu C
O.10 Thus, those three modes are expected to be most affected in the CE-complexed molecule. Indeed, our calculation for the +14.8 kJ mol−1 structure predicts a large blue-shift of the Phe, Gly2, and Leu C
O stretch frequencies that is further confirmed by the experimental results in which these transitions undergo a blue-shift of +55 cm−1, +59 cm−1, and +26 cm−1 respectively. On the other hand, the C
O stretch modes of Gly1 and Tyr, whose H-bonds are not strengthened by the solvation of the charge in the uncomplexed molecule and are most likely less affected by its now reduced interaction with the peptide's backbone, undergo a much minor shift of +3 cm−1 and −4 cm−1, respectively. However, the H-bonding pattern of the 0 kJ mol−1 structure would require the breakage of two additional H-bonds, namely C7 and C14 in Fig. 4b, although none of them plays a significant role in the solvation of the charge and therefore should not be affected by the complexation with a CE.
These results further support the conformational assignment of the +14.8 kJ mol−1 structure based on the experimentally obtained infrared spectrum. Further confirmation could be provided, for example, by measurements in the hydride stretch region, where the calculated spectra of both structures present significant differences. This is, however, beyond the scope of this work. It is also important to note that this structure might not be the global minimum structure and, for example, some rotations of the side groups can not be discarded. Nevertheless, the assigned main H-bond pattern shown in Fig. 4b should not be affected.
The spectra of [Leu-Enk + H]+ shown here can be directly compared to the spectra measured using infrared-ultraviolet (IR-UV) double resonance on [Leu-Enk + H]+ in a cold ion trap10 and to those when performing IRMPD on room temperature [Leu-Enk + H]+.19 In general, there is a very good agreement in peak positions between the helium droplets data and the results obtained using a cold ion trap10 (see Fig. S2, ESI†). The two spectra have very similar peak positions and peak widths. For most transitions, the differences in wavenumber between the helium droplets and IR-UV gas-phase data are smaller than 11 cm−1 and comparable to the widths of the bands themselves. The fact that the peaks in the spectra shown in this work are mostly shifted uniformly in the same direction (towards the red side of the spectrum) might be due to small errors in the wavelength calibration. Only one transition – the deformation of the Tyr ring – has a considerable shift of ≈20 cm−1 with respect to our calculations and the gas-phase data.
A comparison with IRMPD spectra of room temperature [Leu-Enk + H]+19 shows good agreement between 1200 cm−1 and 1600 cm−1. The same number of bands are shown at similar frequencies. However, between 1600 cm−1 and 1800 cm−1, the disagreement between helium droplets and IRMPD data is more evident. For example, the peak measured at 1623 cm−1 in the helium droplets data is absent in the IRMPD data. This is most likely due to the excitation mechanism intrinsic in the IRMPD process where the absorption of the large number of photons is required, which can lead to shifts and broadenings of the spectral lines due to anharmonicities.6 While the absorption of more than one photon is also necessary to eject an ion embedded in a helium droplet, the number of photons required is much smaller than for the IRMPD process. Further, it is likely that after each consecutive absorption in the ≈10 μs long macropulse, the energy is transferred to the helium and the absorption of the next photon will occur from a re-cooled molecule. Therefore, mode anharmonicities are not expected to play a role. The experimental results confirm this, showing well resolved lines that can have a similar bandwidth to that of the laser (≈8 cm−1) for the two investigated molecules. This is also in line with the results reported previously for aniline ions solvated in helium droplets.14
While peak positions match, comparing the relative intensities of the bands of the spectra shown here to the predictions from theory as well as to the spectrum of the cold trap10 relatively large deviations can be found. As more than one photon is required to induce signal, the observed peak intensities non-linearly depend on the laser power. The ejection mechanism of the ion and its dependence on laser power is yet not fully understood. As a first approximation, we here perform a linear correction by dividing the signal by the laser power. Likely, the relative intensities depend on laser power in a more complicated manner and are difficult to use as a measure when comparing experiment to theory.
The high resolution of the IR spectra obtained using helium droplets has been used to further investigate the importance of the charge solvation previously reported by Burke et al.10 In their analysis, the suggested structure of [Leu-Enk + H]+ is supported by seven H-bonds, three of which solvate the –NH3+ group, facilitating a cooperative strengthening of further H-bonds. When a CE molecule is added, its coordination to the –NH3+ group is expected to disrupt those H-bonds. As a result, the H-bonds that were previously strengthened by the charge solvation will become weaker. Indeed, this effect can be observed in the IR spectra of [Leu-Enk + CE + H]+. The three CO oscillators with H-bonds previously strengthened by the solvation of the charge undergo a large blue-shift with respect to the uncomplexed molecule. On the other hand, no essential shift is observed for the two C
O oscillators that were not as strongly influenced by the self-solvation of the charge.
We can conclude that IR excitation of m/z selected peptide ions in helium droplets can be used as a versatile technique to obtain IR spectra of biological molecules. It provides spectra of very cold molecules, does not require the presence of a chromophore, has a rather weak interaction with the helium solvation shell, and can be extended to large species.34
Footnote |
† Electronic supplementary information (ESI) available: Calculated relative energies, additional IR spectra, assignments of vibrational bands. See DOI: 10.1039/c5cp02172c |
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