I. Yudhipratama_ 3rd Year Literature Review Project_Gas-phase spectroscopy_final report

Indra Yudhipratama Page 1 of 16

Gas-phase spectroscopy for conformational study of

polypeptides

Indra Yudhipratama

Department of Chemistry, University of Sheffield, Sheffield S3 7HF, UK.

E-mail: [email protected]

Abstract. Gas-phase spectroscopy has been used for several decades for biomolecules

such as polypeptides. The study in gas-phase spectroscopy reveals the intrinsic

properties of the polypeptides that are commonly hidden in its biological environment.

Besides that, gas-phase spectra of polypeptides can reveal the intramolecular

interactions within the peptide sequence. Dispersion and H-bonding interaction are

mainly investigated in the gas-phase spectroscopy; in some cases salt-bridge

interactions can also be observed. In the gas phase, polypeptides exhibit canonical

structures rather than in zwitterion form. Furthermore, gas-phase spectra of

polypeptides are mainly consistent with the computational calculations which lead to

conformational determination. Thus, it can give a good benchmark for force fields in

computational calculation.

Introduction

The secondary structures of proteins, such as α-helices, β-sheets, or β- or γ-turns,

mainly determine the overall 3-dimensional structures of protein and these structures

are mainly stabilised by interactions such as hydrogen bonding (H-bonding) and

dispersion interactions. Besides that, the interaction with its biological environment also

contributes to the overall structure which means the intrinsic properties of the protein

are hidden. Therefore, to have a better understanding about protein structure, proteins or

mailto:[email protected]


polypeptides must be in isolated condition where no solvent molecules are present. One

of the methods that can be used to study peptides in this isolated condition is gas-phase

spectroscopy.

Gas-phase spectroscopy can provide information about intrinsic properties of the

building block of polypeptide which is commonly hidden in its surrounding [1]. Besides

that, from this spectroscopy the strength of interactions within the polypeptide that give

the 3D structure can also be observed. One of the advantages of gas-phase spectroscopy

is it can give the best data for comparison with theoretical calculations [1]. Furthermore,

the theoretical calculation will be commonly used to account for the spectrum of the

polypeptide. Despite the combination of gas-phase spectroscopy and theoretical

calculations, the study in the gas-phase spectroscopy of polypeptides emphasises the

probing of molecular interaction rather than determining the structure.

This technique implies the polypeptide needs to be brought into gas phase to study

the electronic spectrum of the polypeptide. However, there are some problems to

vaporise the polypeptide in the experimental conditions, which are low vapour pressure

of polypeptide and decomposition under extreme heat.

In this review, the use of gas-phase spectroscopy to study the conformations of

polypeptides will be discussed which will give a better understanding about the

interactions within the polypeptide chains.

Experimental Techniques

Vaporisation of polypeptides

Bringing polypeptides to the gas phase is tricky in experimental conditions due to

low vapour pressure and tendency to decompose during heating. However, there are


many techniques that are commonly used to bring this type of molecule to the gas phase

but these techniques are mainly used in mass spectrometry. One of the methods that can

be used to bring polypeptide into gas phase is laser-desorption jet cooling method.

In this technique, the sample is mixed with graphite powder and applied to a solid

bar such as graphite and then the laser brings the sample, in this case polypeptide, to gas

phase by desorbing from the mixture and cool it down by supersonic expansion with the

noble gase such as He [2] or Ar [3,4] as the carrier gas. Furthermore, the common laser

source in this technique is Nd:YAG laser (1064 nm) [2]. Another method to produce

gas phase polypeptide is by using nanoelectrospray from an acidic solution of volatile

solvents and the molecule is trapped using multipole ion trap. Stearn’s group vaporised

protonated Ala-Tyr and Tyr-Ala dipeptides via nanoelectrospray from 2 x 10-4

M

solutions of dipeptides in 1:1 methanol:H2O solvent and the ions are trapped using

hexapole ion trap [5]. This method is commonly used to study the isolated protonated

peptides which have overall positive charge [5,6].

Electronic spectroscopy

Types of electronic spectroscopy in the study of peptides are double-resonance

spectroscopy such as resonant 2-photons ionisation (R2PI), IR-UV or UV-UV

spectroscopy. These methods are used to explore different energy level of the molecules

using electromagnetic radiation as shown in figure 1.


Figure 1 Diagram from [1] showing of how the energy levels can be explored

using electronic spectroscopy techniques.

With the ionisation process in R2PI technique, it has the advantage of mass

selectivity [1]. Meanwhile in UV-UV or IR-UV, one of the pulses excites the molecule

so the ground state is depleted and the other pulse follows the first pulse as a probe with

a time gap between 50-100 ms [7]. Because the first beam causes a depletion of the

ground state, it is called a burn laser pulse, and due to this depletion a signal decrease of

the probe laser pulse which cause a ‘hole’ in the spectrum; hence UV-UV or IR-UV

spectroscopy is known as spectral-hole burning (SHB) [1]. In IR-UV technique, IR

pulses act as the burn laser pulse to excite vibrational transition which gives the

advantage of this technique to be isomer selective and also can be used in structural

assignment.

Another method to probe the conformation of small peptides or amino acids can be

done by examining X-ray photoemission (XPS) and near edge X-ray absorption fine

structure (NEXAFS) spectra as conducted by Feyer’s group in study of amino acids

alanine and threonine [8] and dipeptides Gly-Gly [9]. These methods probe the

electronic transition within the molecule and the peak-shifting could predict the

interaction within the peptides.


Discussion

The use of laser in gas-phase spectroscopy implies the need of chromophore within

the polypeptides and there are only three amino acids that contain chromophore:

phenylalanine (Phe), tryptophan (Trp), and tyrosine (Tyr) which contains benzene,

indole and phenol respectively as illustrated in figure 2 [1].

Figure 2 Amino acids with chromophore side group.

Tryptophan was used in the first reported study using laser desorption spectroscopy

which investigated the conformation of tryptophan, Trp-Gly, Gly-Trp, and Trp-Gly-Gly

peptide by Cable’s group [2]. This type of amino acids could give more detailed

information about the environment of the chromophore due to their spectra being highly

affected by its surrounding. This high dependency on its environment can be seen in

UV spectrum of amino acids or peptides such as in figure 3. The UV spectrum would

show a vibrational progression which arises from the chromophore and it can reveal its

environment. Then, the interpretation of UV spectrum is mainly extracting the Franck-

Condon activity which is related to the ground states and excited states of the peptides.

A study by Chin’s group in conformational analysis on β-turn motifs in gas-phase small

peptides showed a complex vibrational progression spectrum of Ac-Gly-Phe-NH2

(figure 3c) which implies a distinct structure of the peptides in the ground states relative

to its excited states [4].


Figure 3 UV spectra from [4] of a) Ac-Phe-NH2, b) Ac-Phe-Gly-NH2, and c)

Ac-Gly-Phe-NH2 in the region of Phe due to first π-π* transition.

A similar vibrational progression between Ac-Phe-NH2 and Ac-Phe-Gly-NH2 (figure 3a

and 3b) shows that the environment of benzene ring is similar in those peptides.

However, the presence of chromophore side group could give a computational

calculation challenge as it is difficult to treat stacking interaction that arises from this

chromophore computationally [1].

Gas-phase spectroscopy can provide information about the interactions that stabilise

the secondary structure such as H-bonding interaction. This interaction could give a

signature band in IR spectroscopy where the NH stretching frequency is intense, broad

and shifted to lower frequency (red-shifted). This signature band can also tell how

strong the interaction is as stronger H-bonding interaction could red-shift the NH

stretching frequency significantly. The early study on gramicidin structure in gas phase

relied heavily on this signature band. The spectra reported by Abo-Riziq’s group (figure

4) show a highly red-shifted NH stretching frequency which is consistent with its

helical structure. Besides that, from these spectra it also shows that there is no


H-bonding interaction on OH group of tyrosine which give a sharp band at around

3600 cm-1

[10].

Figure 4 The helical structure and spectra of gramicidin taken from [8].

Řeha’s group demonstrated the strength of H-bonding interaction from the IR spectra

of Phe-Gly-Gly peptides where a less broad and intense peak were observed [3]. This

study enabled the IR spectra to be accounted with the calculated spectra of the global

minimum of Phe-Gly-Gly. It was shown the calculated structure of the global minimum

of the peptides has three weak H-bonding interactions due to the distance being more

than 2 Å [3]. It is noteworthy that the structure of global minimum is similar to the

crystal structure of Phe-Gly-Gly in Cambridge Structural Database (CSD) with the only

difference being no intramolecular H-bonding interaction in the crystal structure [3].

Other structural motifs that are also observed in secondary structures of gas-phase

peptides are β- and γ- turns. Usually, the turning structural motif is formed with the aid

of proline (Pro) or glycine (Gly) amino acids. The used of proline to exhibit turning


structure is due to the pyrrolidine side chain, while steric-free side chain of glycine aids

the formation turning motifs. Besides that, the turning structural motifs are also

stabilised by H-bonding interaction at the backbone.

Study by Chin’s group in the secondary structure of Ac-Phe-NH2 (NAPA),

Ac-Phe-Gly-NH2 (Ac-FG-NH2) and Ac-Gly-Phe-NH2 (Ac-GF-NH2) dipeptides in the

gas phase where NH stretching frequencies were selectively used to account for the

structure [4]. Within this peptides sequences, each amino acid has the local preference

of structural motifs such as Phe prefers βL or γ-turn while Gly prefers γL/D. This study

also used the red-shifted NH stretching frequency to account the strength the H-bonding

interaction. A weakly red-shifted NH stretching frequency in NAPA and Ac-FG-NH2

shows possible C5 H-bonding or NH–π electron of benzene interaction. The density

functional theory (DFT) and optimisation at the B3LYP/6-31+G(d) level shows this

peak arises due to C5 H-bonding interaction which implies the stabilisation of β-turn

motif [4]. On the other hand, C7 H-bonding interaction causes the NH stretching

frequency to be more red-shifted which is observed in the IR spectrum around

3350 cm-1

. This different position of C5 and C7 H-bonding interaction implies C7 is

stronger interaction than C5 which might due to less strained structure of C7 interaction

relative to C5. In Ac-GF-NH2, this intermediate band due to C5 interaction is absent

which means only C7 intramolecular interactions occur in Ac-GF-NH2 and it is

consistent with the calculated spectra of this conformation. This implies Ac-FG-NH2

adopts βL-γ conformation, which is similar to NAPA, while Ac-GF-NH2 adopts γ-γ

conformations [4]. Hence, this study showed that the secondary structure is sequence

dependence. A further study by Alauddin’s group in small peptides sequence containing

either cysteine or serine also shows the local preference of amino acids in turning motifs


as cysteine prefer C5 interaction with SH atom as H-bonding acceptor while serine

prefer C6 interaction with OH group as H-bonding donor [11].

Figure 5 Intramolecular interactions in small peptides.

Another study in small peptide conformation of β- and γ- turns was conducted by

Compagnon’s group which investigated the backbone conformation. This study used a

dipeptide of α-aminoisobutyric acid (Aib) and proline which is protected using

benzyloxycarbonyl (Z) at N-terminal and –NHMe group at C-terminal [12]. This

sequence of peptide is noteworthy as the benzene chromophore is provided by the

protecting group rather than the amino acids. Another point to note in this sequence is

the use of Aib, which is a rare amino acid with 2 methyl groups on α-carbon, to give a

steric strain in the backbone.

In the peptide sequence of Z-Aib-Pro-NHMe, there are 2 possible H-bonding

interactions that can be accommodated in this structure, which are γ-turn and β-turn as

shown in figure 6.


Figure 6 Possible intramolecular interaction in Z-Aib-Pro-NHMe

In this study, the carbonyl stretching frequency was used to probe rather than NH

stretching frequency, which is more useful as shown by the interaction in figure 6. From

the IR spectra of this peptide, three peaks were observed in the region of carbonyl

stretching frequency. The computational calculation aids the assignment of these

carbonyls with C=O close to Z-cap [CO(1)] is at higher frequency relative to the other

bands, the intermediate band is assigned to carbonyl linking C=O close to NHMe

[CO(3)], and the red region carbonyl stretching frequency is assigned to C=O linking

Pro with Aib [CO(2)] [12]. The highly red-shifted band of C=O implies that there is H-

bonding interaction with CO(2), and the blue region of carbonyl stretching shows no H-

bonding interaction with CO(1). This implies Z-Aib-Pro-NHMe exhibits γ-turn and

from the computational calculation the structure exhibit helical structure [12]. This

helical structure is aided by Aib which has strong preference to form helical structure.

However, this study revealed that secondary structure of this peptide in the gas phase is

different to the crystal structure; the crystal structure has β-turn motif [12]. Comparing

this study with study by Řeha’s group earlier, this difference between the condensed

and isolated structure is striking but this study underline the importance of investigation

of biomolecules in the isolated condition. In this phase, the biomolecules are

disentangled from its environment to reveal its intrinsic properties.


Most of the studies of peptides in the gas phase were limited to a small peptide

sequence, mainly a maximum of five amino acids, which is due to increasing the

number amino acids in polypeptide consequently increases the molecular weight. Thus,

it is more difficult to bring the peptides into gas phase. The study in peptide with high

molecular weight was pioneered by a study conducted by Abo-Riziq’s group on the

structure of gramicidin in the gas phase [10]. Gramicidin is a relatively large peptide

with 15 residues with molecular weight between 1845 – 1898 a.m.u. which is larger

than the peptides which had been commonly used. In this study, as discussed earlier, it

was revealed that the gramicidin exhibits helical structure and it was assigned only by

the IR spectroscopy. Further study by Rijs et al. confirmed helical structure of

gramicidin A and C which is consistent with the theoretical calculations and the

experimental spectra [13]. Another study in peptide with high molecular weight was

done by Nagornova’s group on a decapeptide cyclo-VOLFPVOLFP, where “O” is

designated to ornithine, and this decapeptide was probed using cold-ion spectroscopy

which allows vibrational resolution in the UV and IR spectra [14]. The assignment of

this decapeptide was done by assessing the shifting of the NH stretching band and its

computational calculation. Furthermore, molecular dynamic simulation showed a more

compact structure of this decapeptide with H-bonding interaction with π-electron of Phe

chromophore which is absent in the crystallised species [14].

In the gas phase, amino acids and polypeptides are always observed in their

canonical structures, not as zwitterions. This means the study of isolated peptides

always concentrates on H-bonding and π-π dispersion interactions which mainly

stabilise the secondary structure of polypeptides. However, there is another stabilising

interaction that is slightly challenging to study in isolated phase which is salt-bridge

interaction. A study conducted by Rijs’ group showed a zwitterionic structure of the


side chain can be formed in the gas phase [15]. It is noteworthy that the overall charge

is still neutral and to exhibit the zwitterionic structure the peptide needs to have the

most acidic and the most basic side groups which are glutamic acid (Glu) and arginine

(Arg). In this study, Ac-Glu-Ala-Phe-Ala-Arg-NHMe (EAFAR) was used and

phenylalanine which serves as chromophore [15]. The way to observe this zwitterionic

structure was done by comparing the IR spectrum of EAFAR with Z-Glu-OH and Z-

Glu-NH2. The signature band of the unprotonated residue of Glu in the region around

1850 cm-1

which is corresponding to carboxylic acid’s C=O stretching frequency was

used in this study [15].

Figure 7 The IR spectra from [15] of a) capped EAFAR b) Z-Glu-OH and

c) Z-Glu-NH2.


In the spectrum of EAFAR, the absence of a band around 1850 cm-1

indicates the

deprotonation of COOH residue of Glu in the gas phase EAFAR [15]. Besides that, it is

also observed that the stretching frequency of the carbonyl amide is significantly red-

shifted which indicates H-bonding interaction. The conformational search of EAFAR

then conducted by comparing computed and experimental IR spectrum of EAFAR and

it was found the experimental spectrum corresponds to the spectrum of EAFAR global

minimum which shows salt bridge interaction and exhibits helical structure (figure 8).

Figure 8 Diagram from [15] showing calculated low-energy conformations of

zwitterionic EAFAR.

A further study conducted by Jaeqx’s group using Z-Glu-Alan-Arg-NHMe (n = 0, 1,

3) confirmed the salt bridge interaction in gas-phase peptides [7]. On the

conformational search of this peptide, there are 3 possible ways the salt bridge

interaction can be formed as shown in figure 9.

Figure 9 Diagram from [7] showing different types of salt bridge interactions in

conformational search of Z-Glu-Alan-Arg-NHMe.


Jaeqx’s group used the COO– symmetric carbonyl stretching frequency to observe

this interaction due to its sensitivity to the environment. Another interesting feature

from Z-Glu-Alan-Arg-NHMe peptides is the Arg residue also interacts with benzene

ring of the Z-cap via NH-π electrons interaction; the exact interaction between Arg

residue and π-electrons of benzene is different between each system. This study shows

that all systems exhibit the A-type interactions and this is also observed in the previous

study. Furthermore, Jaeqx’s group also noted that B-type interaction is possible to

observe when Glu and Arg residues are further apart and it might be due less strain on

the backbone as the peptide becomes longer [7].

Conclusions

In the past decades, gas-phase spectroscopy has been used to investigate

biomolecules, such as polypeptides, in its isolated phase. These studies give several

methods to bring biomolecules into gas phase such as laser desorption and electrospray

techniques which are then coupled with laser spectroscopy.

The studies in the gas-phase spectroscopy of polypeptides reveal the intrinsic

properties of the polypeptides such as the intramolecular interactions within the peptide

sequence. Mainly, the secondary structure of peptides in the gas phase is stabilised by

H-bonding and dispersion interactions. Several studies also started to look at the

possibility of salt-bridge interactions in the isolated condition by using Glu and Arg in

the peptides due to Glu and Arg has the most acidic and basic residue respectively. The

gas-phase spectroscopy is mainly used to investigate the intramolecular interactions in

the peptides rather than determination of the secondary structure. This conformational

search of secondary structure is aided by computational calculation which consistently

shows the agreement between theoretical calculation and experimental spectra. Thus,


the intrinsic structural motifs of the polypeptides can be revealed and commonly the

structure resembles in its crystallised form or in its biological environment.

Furthermore, this shows the advantage of gas-phase spectroscopy that can be used as a

benchmark for force fields in computational calculation.

References

1 M. S. de Vries and P. Hobza, “Gas-Phase Spectroscopy of Biomolecular Building

Blocks”, Annu. Rev. Phys. Chem., 2007, 58, 585-612.

2 J. R. Cable, M. J. Tubergen, and D. H. Levy, “Laser desorption Molecular Beam

Spectroscopy: The Electronic Spectra of Tryptophan Peptides in the Gas Phase”, J. Am.

Chem. Soc., 1987, 109, 6198-6199.

3 D. Řeha, V. Valdés, J. Vondrášek, P. Hobza, A. Abu-Riziq, B. Crews, and M. S. de

Vries, “Structure and IR Spectrum of Phenylalanyl-Glycyl-Glycil Tripeptide in the Gas-

Phase: IR/UV Experiments, Ab Initio Quantum Chemical Calculations, and Molecular

Dynamic Simulations, Chem. Eur. J., 2005, 11, 6803-6817.

4 W. Chin, J. P. Dognon, F. Piuzzi, B. Tardivel, I. Dimicoli, and M. Mons, “Intrinsic

Folding of Small Peptide Chains: Spectroscopic Evidence for the Formation of β-Turns

in the Gas Phase”, J. Am. Chem. Soc., 2004, 127, 707-712.

5 J. A. Stearns, M. Guidi, O. V. Boyarkin, and T. R. Rizzo, “Conformation-specific

infrared and ultraviolet spectroscopy of tyrosine-based protonated dipeptides”, J. Chem.

Phys., 2007, 127, 154322-1 – 154322-7.

6 J. A. Stearns, O. V. Boyarkin, and T. R. Rizzo, “Spectroscopic Signatures of Gas-

Phase Helices: Ac-Phe-(Ala)5-Lys-H+ and Ac-Phe-(Ala)10-Lys-H

+”, J. Am. Chem. Soc.,

2007, 45, 13820-13821.


7 S. Jaeqx, J. Oomens, and A. Rijs, “Gas-phase salt bridge interactions between

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8 V. Feyer, O. Plekan, R. Richter, M. Careno, K. C. Prince, and V. Carravetta, “Core

Level Study of Alanine and and Threonine”, J. Phys. Chem. A, 2008, 112, 7806-7815.

9 V. Feyer, O. Plekan, R. Richter, M. Careno, K. C. Prince, and V. Carravetta,

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J. Phys. Chem. A, 2009, 113, 10726-10733.

10 A. Abo-Riziq, B. O. Crews, M. P. Callahan, L. Grace, and M. S. de Vries,

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11 M. Alauddin, H. S. Biswal, E. Gloaguen, and M. Mons, “Intra-residue interactions

in proteins: interplay between serine or cysteine side chains and backbone

conformations, revealed by laser spectroscopy of isolated model peptides”, Phys. Chem.

Chem. Phys., 2015, 17, 2169-2178.

12 I. Compagnon, J. Oomens, G. Meijer, and G. von Helden, “Mid-Infrared

Spectroscopy of Protected Peptides in the Gas Phase: A Probe of the Backbone

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13 A. M. Rijs, M. Kabeláč, A. Abo-Riziq, P. Hobza, and M. S. de Vries, “Isolated

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14 N. S. Nagornova, M. Guglielmi, M. Doemer, I. Tavernelli, U. Rothlisberger, T. R.

Rizzo, and O. V. Boyarkin, “Cold-ion Spectroscopy Reveals the Intrinsic Structure of a

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I. Yudhipratama_ 3rd Year Literature Review Project_Gas-phase spectroscopy_final report