# The relationship between the tyrosine residue 850-830 cm⁻¹ Raman doublet intensity ratio and the aromatic side chain χ 1 torsion angle

Belén Hernández, Yves-Marie Coïc, Sergei Kruglik, Santiago Sanchez-Cortes, Mahmoud Ghomi

► To cite this version:

Belén Hernández, Yves-Marie Coïc, Sergei Kruglik, Santiago Sanchez-Cortes, Mahmoud Ghomi. The relationship between the tyrosine residue 850-830 cm⁻¹ Raman doublet intensity ratio and the aromatic side chain χ 1 torsion angle. Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy [1994-..], 2024, 308, pp.123681. 10.1016/j.saa.2023.123681. hal-05090580

HAL Id: hal-05090580

https://hal.science/hal-05090580v1

Submitted on 30 May 2025

**HAL** is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
The relationship between the tyrosine residue 850-830 cm⁻¹ Raman doublet intensity ratio and the aromatic side chain χ₁ torsion angle

Belén Hernándezª, Yves-Marie Coïcᵇ, Sergei G. Kruglikᶜ, Santiago Sanchez-Cortesᵈ,
Mahmoud Ghomiᵈ*

ªLVTS, INSERM U1148. 74 rue Marcel Cachin. 93017 Bobigny Cédex France

ᵇInstitut Pasteur, Université Paris Cité, CNRS UMR3523, Unité de Chimie des Biomolécules, F-75015 Paris, France

ᶜSorbonne Université, CNRS, Institut de Biologie Paris-Seine, Laboratoire Jean-Perrin, 4 Place Jussieu, 75005 Paris, France

ᵈInstituto de Estructura de la Materia, IEM-CSIC, Serrano 121, 28006 Madrid, Spain

*Corresponding author: M. Ghomi, E-mail: [mahmoud.ghomi@univ-paris13.fr](mailto:mahmoud.ghomi@univ-paris13.fr)
ABSTRACT

Tyrosine (Tyr) residue in a peptide chain is characterized by the presence of seven Raman
markers, referred to as Yᵢ (i=1, ..., 7), distributed over the middle wavenumber spectral
region. Particularly, the changes observed in the relative intensity of Y₅ and Y₆ markers,
appearing as a side by side doublet at *ca.* 850-830 cm⁻¹, has received a great attention.
Primarily assigned to a Fermi-resonance effect between phenol ring planar and nonplanar
modes, former density functional theory calculations led us to affiliate the Y₅-Y₆ doublet to
two distinct fundamental modes. Furthermore, despite the previous assumptions, it was
evidenced that the reversal of the doublet intensity ratio cannot be solely explained by
hydrogen bonding on the phenol hydroxyl group involved in Tyr. Herein, upon analyzing the
observed and theoretical data collected from the cationic species of the tripeptide Gly-Tyr-
Gly, the crucial effect of the aromatic side chain orientation, especially that of the χ₁ torsion
angle defined around the Cα-Cβ bond, on the Tyr doublet intensity ratio has been evidenced.

**Keywords:** Peptide; Glycyltyrosylglycine; Extended chain; Polyproline-II; Helix; γ-turn; Raman spectra, DFT calculations
# 1. Introduction

Aromatic residues provide characteristic Raman lines in the vibrational spectra recorded from peptides and proteins [1-18]. This is due to the strong modulation of the electronic polarizability by the atomic displacements relative to certain vibrational motions occurring in the phenyl, phenol, indole and imidazole rings involved in phenylalanine (Phe), tyrosine (Tyr), tryptophan (Trp) and histidine (His) side chains, respectively [19].

We focus here on the Tyr characteristic Raman lines [13,17,18]. In a former joint experimental and theoretical analysis, it was mentioned that Tyr gives rise to seven Raman markers, referred to as $Y_i$, $i=1, ..., 7$, distributed throughout the middle wavenumber region (1800-600 cm⁻¹), and observed as strong lines located at ~1616 (Y₁), ~1606 (Y₂), ~1210 (Y₃), ~1178 (Y₄), ~850 (Y₅), ~830 (Y₆), and ~643 (Y₇) cm⁻¹ [18]. Since the earliest vibrational studies on peptide chains, the behavior of the pair Y₅-Y₆, observed as a side by side doublet in Raman spectra, has received attention. The influential work reported by Siamwiza et al. in 1975 [2], assigned the Tyr doublet to a Fermi-resonance effect. Precisely, on the basis of the Raman scattering observations accompanied by a normal mode analysis on a simple model compound, p-cresol (para-methylphenol), the authors claimed that Y₅ might be arising from a Raman-active planar mode, whereas Y₆ results from the interaction (Fermi-resonance) of Y₅ with the first overtone of an IR-active out-of-plane mode at ~413 cm⁻¹, having a wavenumber at ca. 2x413=826 cm⁻¹. In this explanation, it was admitted that because of the symmetrical reasons the overtone of an IR-active mode becomes Raman-active. This interpretation cannot unfortunately hold upon considering the p-cresol→Tyr symmetry lowering caused by the connection of the phenol ring to two successive side chain bonds $C_γ-C_β$ and $C_β-C_α$ (Fig. 1). As known, Tyr residue belongs to $C_1$ symmetry, in which all vibrational modes are Raman- and IR-active. Consequently, the p-cresol-based vibrational selection rules lose their validity in a structurally more complex molecular entity such as Tyr. Density functional theory (DFT)
calculations on free amino acid (Tyr) with zwitterionic amine/acid terminal groups $(NH_3^+/COO^-)$ [18,20,21], proved that the doublet arises from two distinct fundamental modes with a predominant planar character for $Y_5$, assigned to a ring breathing motion, and an out-of-plane nature for $Y_6$, mainly arising from the C-H wagging motions.

On the other hand, based on several classical and ultraviolet Raman scattering studies [2-10], it appeared that although the wavenumbers of $Y_5$ and $Y_6$ manifests a limited variation (±5 cm⁻¹), their intensity ratio, $ρ = I_{850} / I_{830}$ (where I is the Raman intensity), shows a large amplitude of variation (0.3<ρ<2.5). It had been thought that the lowest ρ value is observed when the phenol OH group acts as a H-bond donor, whereas its highest value corresponds to the hydroxyl group playing the role of a H-bond acceptor. By extension, an intermediate ρ value is expected when hydroxyl acts as both H-bond donor and acceptor. Unfortunately, this assumption has not been validated by the DFT calculations on a zwitterionic Tyr explicitly hydrated by water molecules [18], which conformed that H-bonding cannot explain the ρ value reversal.

The insufficiency of simple molecular models, such as p-cresol [9,18] and free amino acid (Tyr) [18,20,21], to accurately interpret the observed variation of ρ, led us to the conclusion that this phenomenon might be conformational. In this condition, a molecular compound including a Tyr residue and a larger backbone size was required for a more complete conformational investigation. However, it became rapidly evident that a dipeptide model would also be insufficient, simply because of its $N^{\text{ter}}/C^{\text{ter}}$ truncated structure. Therefore, we attempted to consider a tripeptide model with the primary sequence Gly-Tyr-Gly (Fig. 1) because (i) it includes two peptide (-CO-NH-) bonds located at either side of the middle residue, rendering possible a more accurate analysis of their coupling, as well as the nonbonded interactions occurring between the Tyr side chain and one of its neighbouring peptide bonds; (ii) the two Gly residues present limited and localized modes [22], which
would not be superimposed to the characteristic Tyr residue characteristic Raman lines; (iii) the backbone polymorphism of tripeptides has also been subject to several studies. For instance, it has been evidenced that despite its short length, a tripeptide is not disordered in aqueous media. In this context, the application of optical spectroscopic data based on a phenomenological formalism [23,24] led to the assessment of ordered backbone conformations, such as β-strand, polyproline II (pP-II), helical, and γ-turn folds in aqueous samples of tripeptides [25]. Particularly, in the case of zwitterionic Gly-Tyr-Gly, this methodology had confirmed the presence of pP-II conformers coexisting with other possible secondary structures [26]; (iv) although the room temperature classical Raman spectrum of zwitterionic Gly-Tyr-Gly recorded at 20 mM, pH7, was available [17], its accompanying DFT calculations performed at the B3LYP/6-311+G(2df,2pd) level was based on only one conformer resembling somehow an extended chain (see Section 3.2 for details). This fact also justified a more complete multiconformational analysis of the tripeptide.

Taking benefit from our recent data on the conformational and vibrational studies of tripeptides [22,27], our aim was to achieve a better understanding of the influence of intramolecular interactions, especially those induced by the side chain orientation and backbone conformation, on the Y<sub>5</sub>-Y<sub>6</sub> doublet relative intensity.

**2. Experimental and theoretical methods**

2.1. *Chemical synthesis of tripeptide*

The tripeptide NH₂-Gly-Tyr-Gly-CONH₂, having amine/amide terminal groups (Fig. 1), was synthesized at the Institut Pasteur (Paris, France) according to the Fmoc/tBu solid-phase strategy [28] from a Rink amide resin on an ABI 433 synthesizer (Applied Biosystems, Foster City, CA). Cleavage from the solid support was accomplished by treatment with a 95:2.5:2.5 mixture of trifluoroacetic acid (TFA), triisopropylsilane (TIS), and water for 2:30 h at room
temperature. After filtration of the resin, the cleavage mixture was poured into ice-cold diethyl ether. The precipitate was recovered by centrifugation, washed three times with diethyl ether, dried, dissolved in aqueous TFA, and freeze dried. The crude product was solubilized in aqueous TFA 0.08% and purified on reverse phase by medium-pressure liquid chromatography (AP-100/200 flash, Armen Instrument, Saint Ave) using a preparative column (26×313mm) packed with 100-Å 20-µm C18 Nucleoprep packing (Macherey & Nagel GmbH & Co., Düren), by applying a linear gradient of solvent B (mixture of MeCN and solvent A, 8 : 2 v/v) in solvent A (0.08% aqueous TFA, pH 2) over 60 min at a 20 mL/min flow rate. Proper fractions were pooled and lyophilized. The final net peptide content was determined by quantitative amino acid analysis, giving an overall isolated yield of 81% for the tripeptide. The purity control of the final peptide was performed by RP-HPLC (Reversed-phase High-performance liquid chromatography) on an Agilent (Santa Clara, CA) 1100 series liquid chromatograph and monitored by absorbance at 230 nm, by applying a linear gradient of acetonitrile in 0.08% aqueous TFA over 20 min at a 0.35 mL/min flow rate on an Aeris Peptide 3.6 µm XB-C18 analytical (2.1×100 mm) column (Phenomenex, Le Pecq). Electrospray ionization mass spectrometry was carried out on a quadrupole-TOF Micro mass spectrometer (Waters) equipped with a Z-spray API source. The experimental mass was acquired in positive-ion mode and was consistent with the theoretical isotopic value (expected M+Na⁺ 317.1226, observed 317.0705).

## 2.2. Samples for Raman spectroscopy

Lyophilized powder samples of tripeptide were dissolved in water taken from a Millipore filtration system. The final concentration of the aqueous solution was 20 mM (~6 g/L) providing a good signal/noise ratio. Upon dissolution, the pH value of the tripeptide samples was 6.5±0.1. In this condition the amine terminal group is protonated (pKₐ~10.5). Therefore, the presented Raman spectra correspond to the cationic species of the tripeptide with
$NH_3^+/CONH_2$ backbone terminal groups (Fig. 1). H-D isotopic substitution on labile hydrogen atoms involved in NH and OH chemical bonds, was achieved by dissolving the compound in $D_2O$ (100% purity) purchased from Eurisotop (Saclay, France).

Samples containing 50 µL of the prepared aqueous solution were placed in a suprasil quartz cell (5-mm path length) and excited by the 488 nm line of an $Ar^+$ laser (Spectra Physics), ~200-mW power at the sample. Scattered light at right angle was analyzed on a T64000 (HORIBA Jobin Yvon, Longjumeau, France) in a single spectrograph configuration with a 1200 groove per millimeter holographic grating and a holographic notch filter. Stokes Raman data were collected by means of a liquid nitrogen-cooled charge-coupled device detection system (Spectrum One, Jobin Yvon). The effective spectral slit width was set to ~5 $cm^{-1}$. An average accumulation time of 1200 s (40 scans of 30 s each one) was chosen for recording the reported Raman spectra. GRAMS/32 Z.00 (Thermo Galactic, Waltham, MA, United States). The final presentation of these spectra was performed by means of SigmaPlot package 6.10 (SPSS Inc., Chicago, IL, United States).

## 2.3. Initial rotamers of backbone and side chain for DFT calculations

The backbone conformation of the tripeptide (Fig. 1A) is governed by seven torsion angles ($\psi_1, \omega_1, \Phi_2, \psi_2, \omega_2, \Phi_3, \psi_3$) defined around the successive chemical bonds in going from N'ter to C'ter side [19], where the subscripts recall the number of residues (Gly¹, Tyr² and Gly³). While $\omega$ remains close to 180°, the values of the ($\Phi, \psi$) angles depend on the backbone secondary structure. Herein, we only limit our conformational analysis to ordered structures (see Section 1 for details), whose initial rotamers were prepared using the backbone torsion angles ($\Phi, \psi$)=(-135°,+135°) for β-strand, ($\Phi, \psi$)=(-75°,+150°) for pP-II, and ($\Phi, \psi$)=(-60°,-45°) for α-helix. There are two γ-turn structures, appearing as mirror images, referred to as classic and inverse γ-turn [29,30]. In
both cases, the folds were supposed to be centered on the middle residue (Tyr²). The initial
values given to the (Φ₂,ψ₂) torsion angles were: (Φ₂,ψ₂)=(+75°,-65°) for classic γ-turn and
(Φ₂,ψ₂)=(-75°,+65°) for inverse γ-turn. All other backbone torsion angles of a γ-turn
conformer, i.e., those relative to Gly¹ and Gly³ residues, were initially set to the values of
corresponding to a β-strand.

Two additional torsion angles, namely (χ₁,χ₂), defined around the two successive Tyr² side
chain bonds (Fig. 1B), are required to account for its conformational flexibility [13,18,19]. As
Cα and Cβ atoms are of sp³ type, three privileged orientations, referred to as gauche⁺ (g⁺)
(+60°±60°), gauche⁺ (g⁻) (-60°±60°), and trans (or t) (180°±60°), can be assigned to the
torsion angle defined as χ₁(N-Cα-Cβ-Cγ). The initial values of χ₁ were +60°, -60° and 180°
corresponding to its g⁺, g⁻ and t orientations, respectively. As far as χ₂ is concerned, because
of the equivalence of Cδ₁ and Cδ₂ atoms in phenol ring, there are two manners to define χ₂,
either as χ₂(Cα-Cβ-Cγ-Cδ₁) or as χ₂(Cα-Cβ-Cγ-Cδ₂) (Fig. 1B), one of them is positive (g⁺) and
the other negative (g⁻), and the absolute value of their difference is ~180°. However, these two
angles can be distinguished one from another by the orientation of the phenyl hydroxyl group
(Oη-H) (Fig. 1A&B). It is to be noted that the most energetically favorable locations of Oη-H
are in the phenol plane (not detailed here). In other words, the Oη-H location in phenol ring
can also precise the g⁺/g⁻ orientation of χ₂. Note also that χ₂ is determined by the rotation
around the side chain Cβ-Cγ bond, where Cγ is of sp² type. The privileged orientations of χ₂
are thus g⁺ (90°±90°) and g⁻ (-90°±90°) [13,16,18,27]. The initial values given to χ₂ angle
were +90° (g⁺) and -90° (g⁻).

Considering all the mentioned points, six possible Tyr side chain rotamers were
considered, referred to as g⁻g⁻, g⁻g⁺, g⁺g⁻, g⁺g⁺, tg⁻ and tg⁺, where the first and second letters to
χ₁ and χ₂ orientations, respectively. As a whole, with the five considered backbone
conformers and the six side chain rotamers, 5x6=30 initial conformers were used in each
series of theoretical calculations by means of a given functional.

2.4. *Functionals, basis sets and hydration model used in DFT calculations*

As known, in the theoretical investigations on structural and vibrational features of
peptide chains, numerous effects such as nonbonded interactions, H-bonding, charge transfer
and dipole interactions, should be considered. These effects can only be addressed by
quantum mechanical calculations. As far as the DFT approach [31] is concerned,
sophisticated exchange and correlation functionals have been under development during the
last years with the aim of considering as correctly as possible the noncovalent effects in
molecular systems [32]. In a recent work on the structural and vibrational analysis of the
tripeptide (Gly)₃ [22], eleven functionals of different types (B3LYP, CAM-B3LYP, B97,
B97-1, B97-2, B97-D3, B98, ωB97XD, PBE0, APF-D, M062X and MN15) were tested
through the comparison of their performances with those relative to the Møller-Plessett
second-order perturbation (MP2) method [33]. It appeared that ωB97XD [34-36] was the only
functional capable of providing acceptable data for both cationic and zwitterionic species.
However, the main difficulty encountered in DFT calculations concerned the exaggerated
values of relative energies compared to those predicted by MP2. On the other hand, a former
work [27] on the cationic species of four tripeptides showed that M062X functional [37] can
alleviate certain deficiencies of the widely used B3LYP functional [38,39]. As a result, in the
present work we attempted to compare the performances of three functionals of different
types, B3LYP, M062X and ωB97XD, in the DFT calculations on the Gly-Tyr-Gly cationic
species.
Polarized triple-zeta Gaussian atomic basis sets, equipped with diffuse functions, referred to as 6-311++G(d,p), were used. To test the reliability of the calculated data, AUG-cc-pVTZ basis sets were also employed in certain cases (see Section 2.5 for details).

Hydration effects were considered by a purely implicit model, which consists in placing the solute in a polarizable continuum medium (PCM) [40,41] with a relative permittivity corresponding to bulk water (ε<sub>r</sub>=78.39).

2.5. *Geometry optimization, energy order and Raman intensities of conformers*

DFT calculations were performed using the Gaussian16 package [42]. Geometry optimization on each conformer was followed by harmonic vibrational calculations. The absence of any imaginary frequency confirmed the correspondence of an optimized geometry to a local energy minimum. The energy order of the optimized geometries was based on their total energy (E<sub>tot</sub>), which is the sum of electronic energy (E<sub>e</sub>) and free energy correction (ΔG), i.e., E<sub>tot</sub>=E<sub>e</sub>+ΔG. Each optimized conformer is characterized by its relative energy (ΔE) as compared to that of the lowest energy conformer, for which ΔE was set to zero. In the case of the calculations by means of ωB97XD, two series of relative energies (referred to as ΔE and ΔE') were calculated, where ΔE was completely estimated by means of E<sub>e</sub> and ΔG values using 6-311++G(d,p) basis sets, and ΔE' was determined by single point calculations on the electronic energy (E<sub>e</sub>) using AUG-cc-PVTZ basis sets, and the ΔG values previously obtained by means of 6-311++G(d,p) basis sets.

Raman activities issued from DFT calculations were converted to Raman intensities
following the previously reported expression [43]. Assignment of the vibrational modes was
made possible by the use of the calculated force constants and atomic displacements, leading
to the evaluation of the potential energy distribution (PED) [44], as expressed in terms of
internal coordinates, i.e., bond stretching (v), angular bending (δ), out-of-plane bending (ω), and torsion (τ) coordinates.

# 3. Results and discussion

## 3.1. Raw Raman spectra and their post-record treatment

To accurately estimate the relative intensities of the Raman bands in the middle wavenumber spectral region (1800-600 cm⁻¹), in which backbone and side chain vibrational markers are observed, the buffer background should be thoroughly treated. Traces A and B in Fig. 2 show the raw Raman spectrum obtained from the tripeptide solution and its solvent (H₂O), respectively. We have previously shown how the resolved and well separated bands arising from TFA (counterion arising from the tripeptide chemical synthesis, see Section 2.1 for details) can be easily subtracted [45]. It should be noted that the most intense TFA band is observed at ~1437 cm⁻¹ (shown by a red asterisk in Fig. 2A). The whole background treatment (from TFA and H₂O buffer), gives rise to the spectrum displayed in Fig. 2C. In the same manner, the subtraction of the background from the Raman signal recorded in D₂O buffer leads to the spectrum shown in Fig. 2D (details not shown).

Fig. 2C allows us to appreciate the seven Tyr side chain markers (Y₁, ..., Y₇). Their wavenumbers are similar to those previously observed in the zwitterionic species of the tripeptide [17]. The wavenumber downshifts of these markers do not exceed 2 cm⁻¹ upon selective deuteration (Fig. 1D). However, the most considerable effect upon deuteration is related to the doublet Y₅-Y₆, whose intensity ratio (ρ) varies from ρ~2 (in H₂O, Fig. 1C) to ρ~1 (in D₂O, Fig. 1D). Due to the short length of the analysed peptide, the amide I signal (assigned to the backbone C=O bond-stretch motions) is observed as a weak band at ca. 1693 cm⁻¹ (Fig. 1C), showing a ~10 cm⁻¹ downshift upon deuteration (Fig. 1D).
## 3.2. Deficiencies of functionals used in DFT calculations

The main difficulty encountered in the course of full geometry optimization of the conformers was basically related to γ-turn structures (see Section 2.2 for details). Although we succeeded in obtaining acceptable optimized conformers for classic γ-turn conformers by means of B3LYP and ωB97XD, both functionals failed to maintain the stability of inverse γ-turn conformers. In fact, the conservation of the $g^+$ orientation of $ψ_2$ torsion angle (characteristic of an inverse γ-turn structure) appeared to be impossible in a full geometry optimization. The situation was even more critical with M062X functional because of its failure to provide a complete set of optimized conformers either for classic or for inverse γ-turn structures. The reason of this impossibility was also due to maintaining $ψ_2$ angle either in $g^+$ or in $g^-$ orientation corresponding to classic or inverse γ-turn structures, respectively. However, as we know from our previous calculations on cationic tripeptides [22,27], classic and inverse γ-turn conformers form the highest energy structures. Therefore, the deficiencies encountered with the used functionals to optimize certain γ-turn structures cannot be considered as an obstacle for finding the low energy conformers required in the rest of our discussion (vide infra).

## 3.3. Energy order, conformational angles and intramolecular hydrogen bonds

Tables 1-3 show the relative energies, as well as the conformational angles of the conformers obtained upon full geometry optimization (see Section 3.2 for details). Instead of reporting the optimized conformers in the increasing/decreasing order of their relative energies, we have chosen to keep together the conformers relative to the same secondary structure. Furthermore, for a given backbone secondary structure, the six conformers related to the Tyr side chain rotamers were displayed in the same order: $g^-$, $g^-$, $g^+$, $g^+$, $g^+$, $g^+$, $g^-$ and
$\text{tg}^+$ (see Section 2.2 for details), facilitating the comparison of their energetic and conformational data. To clarify our discussion, the graphical representations of the conformers are shown in Figs. 3-6.

The common features of the theoretical data can be summarized as follows (i) a conformer with a β-strand initial structure was systematically transformed into a quasi-planar structure (Fig. 3), in which except for the couple ($\Phi_2, \psi_2$) relative to Tyr$^2$ the absolute values of other ($\Phi, \psi$) torsion angles (belonging to Gly$^1$ and Gly$^3$) are close to 180° (Tables 1-3). This type of secondary structure was referred to as “extended chain” since our recent works on tripeptides [22,27]; (ii) as expected, whatever the functional, B3LYP (Table 1) or $\omega$B97XD (Table 3), classic $\gamma$-turn conformers form the highest energies ones; (iii) for a given backbone conformation, the change in the relative energy induced by the side chain conformation mainly arises from the $\chi_1(\text{g}^-, \text{g}^+)$ and $\text{t}$ orientation. In a general manner, the $\chi_2(\text{g}^- \leftrightarrow \text{g}^+)$ orientation change presents a low energy cost, i.e., located below 0.8, 0.4, and 0.5 kcal/mol, as predicted by means of B3LYP, M062X and $\omega$B97XD functionals, respectively (Tables 1-3). Even if these energy costs are more or less exaggerated, as evidenced by the previous calculations on (Gly)$_3$ [22], they however indicate that at room temperature (RT~0.6 kcal/mol) both right and left orientations of phenol hydroxyl can coexist (Fig. 1A&B, see also Section 2.3 for details). In other words, for a given $\chi_1$ orientation ($\text{g}^-, \text{g}^+$ and $\text{t}$), both sister conformers relative to $\text{g}^-$ and $\text{g}^+$ orientations of $\chi_2$, can be simultaneously present in aqueous environment in normal conditions.

The functional type has a considerable effect on the energy landscape (Tables 1-3) of the tripeptide. For instance, B3LYP and $\omega$B97XD place the lowest energy conformers ($\Delta E=0$) among extended chain conformers, but with different side chain orientations, i.e., $\text{tg}^-$ (for B3LYP, Table 1) and $\text{g}^+\text{g}^+$ (for $\omega$B97XD, Table 3). Surprisingly, the lowest energy conformer provided by M062X is a helical one having a $\text{g}^+\text{g}^+$ side chain orientation (Table 2). At this
step, it seemed interesting to check whether the energy order can depend on the basis sets.
The last column of Table 3 displays the relative energies (ΔE') obtained using AUG-cc-PVTZ
basis sets together with ωB97XD functional (see Section 2.5 for details). Apart from some
changes in the values of relative energies, any remarkable difference has been encountered
neither in the conformational nature of low energy conformers, nor in the global energy order
of different secondary structures.

As shown in Figs. 4 and 6, helical and classic γ-turn conformers are characterized by an
intramolecular H-bond, of which the length (d<sub>HB</sub>) are reported in Tables 1-3. In a helical form,
this H-bond, playing somehow the role of the so-called i→i+4 H-bond in an α-helix
[19,22,27], is formed between the backbone C=O (Gly<sup>1</sup>) and one of the hydrogen atoms
involved in the C<sup>ter</sup> amide group (Fig. 4). In helical conformers, d<sub>HB</sub> was shown to be
functional dependent, located in the ranges 2.15-2.28 Å (Table 1), 2.05-2.15 Å (Table 2) and
2.07-2.13 Å (Table 3) using B3LYP, M062X and ωB97XD, respectively. An intramolecular
H-bond in classic γ-turn (also known under the name “closing H-bond”) is formed between
the backbone C=O (Gly<sup>1</sup>) and N-H (Gly<sup>3</sup>) (Fig. 6). This nonbonded interaction is
characterized by a shorter length (d<sub>HB</sub>), located in the intervals 1.83-2.10 Å (Table 1) and
1.80-2.05 Å (Table 3), as predicted by B3LYP and ωB97XD functionals, respectively.

3.4. Scaling calculated vibrational wavenumbers

In the recent work devoted to (Gly)₃ [22], the advantage of using an affine scaling
equation of the form: vscal=avcalc+b (where vscal and vcalc represent scaled and raw
wavenumbers), was highlighted. In this equation, the constant (a) is in fact the widely used
(multiplying) scale factor employed by many authors to correct the calculated wavenumbers.
The use of the constant (b) was shown to be necessary, simply because the scaling procedure
is region dependent. More precisely, while the wavenumbers >900 cm⁻¹ can be approximately
scaled by just one constant (a), those <900 cm⁻¹ are located close (or even below) the calculated ones, requiring a positive constant (b) to be correctly scaled.

Graphs A-C in Fig. 7 show the application of the linear scaling on the vibrational modes calculated by means of the three selected functionals. In these graphs, the circles possess the coordinates: $v_{\text{obs}}$ (an observed wavenumber taken from Fig. 2C) along the vertical axis, and $v_{\text{calc}}$ (the corresponding calculated wavenumber) along the horizontal axis. The solid lines in these graphs are issued from a linear regression, of which the constants (a,b) are also reported on each graph. The correlation coefficient (r) in all cases was greater than 0.995 in all cases.

It is noteworthy that B3LYP gives the closest wavenumbers to the observed ones (Fig. 7A), as perceived by the values of linear constants (a=0.9836, b=4.115 cm⁻¹). While the constant (a) gradually decreases when M062X (Fig. 7B) and ωB97XD (Fig. 7C) are employed, the constant (b) increases (Fig. 7B&C). The conclusion is that the calculated wavenumbers using B3LYP and ωB97XD present the smallest and the largest differences, respectively, with respect to the observed wavenumbers.

### 3.5. Focus on the calculated Raman spectra in Tyr doublet region

The question raised here was to see whether the backbone and side chain conformations affect the Tyr doublet intensity ratio (ρ). It should be recalled that because of the phenol ring constitution, the ring breathing motion (planar), as well as the C-H wagging motions (out-of-plane), occur within (or close to) the 900-800 cm⁻¹ region. As the four C-H bonds are symmetrically located in the six membered phenol ring, they participate in (two by two) in-phase and out-of-phase motions (not detailed here).

In Figs. 3-6, for each backbone conformation (extended chain, helical, pP-II and classic γ-turn), the calculated Raman spectra are displayed. Each Figure is subdivided into three segments (A, B and C), revealing the effect of the side chain orientation on the calculated
Raman spectra: g$^-g$ and g$^+g$ (segment A), g$^+g$ and g$^-g$ (segment B), tg$^-$ and tg$^+$ (segment C).
For a given backbone conformation and a given side chain orientation, apart from the
differences in their peak wavenumbers (see Section 3.4 for details), the spectral shape
(number of bands and their relative intensities) is shown to be independent of the used
functional. Moreover, for a given $\chi_1$ (g$^+$, g$^-$ or t) orientation, g$^+$ and g$^-$ orientations of $\chi_2$
provide similar spectral shapes (Segments A-C, Figs. 3-6). In Section 3.3 we have mentioned
that the $\chi_2$ (g$^-\leftrightarrow$g$^+$) side chain orientation change is in all cases accompanied by a small
energy cost. At last, while the side chain $\chi_1$ orientation shows a considerable effect on the
doublet, the backbone conformation preferentially affects the relative intensities of the
satellite bands in this region, i.e., those mainly arising from the C-H wagging motions.

### 3.6. Calculated Raman spectra in the middle wavenumber spectral region

Previous works devoted have shown that the observed Raman spectrum from amino acids and peptides [16,18,46,47] can be assumed as the thermal (Boltzmann) average of the spectra arising from their low energy conformers. In this context, the lowest energy conformer (corresponding to $\Delta E=0$) brings naturally the most important contribution to Raman intensity.
As mentioned in Section 3.3, the calculated energy landscape of the tripeptide is clearly functional dependent (Tables 1-3). To find the functional type providing the most accurate energetic and vibrational data. The response to this question can be given upon the comparison between the calculated and observed Raman spectra. In Fig. 8 the observed Raman spectrum (Trace A) is compared to those calculated with B3LYP (Trace B), M062X (Trace C), and ωB97XD (Trace D) functionals. Considering the energetic criteria detailed in Section 3.5, each calculated spectrum is the thermal average of two calculated Raman spectra arising from the lowest energy conformer (corresponding to $\Delta E=0$), and its energetically sister conformer obtained by the $\chi_2$ (g$^-\leftrightarrow$g$^+$) orientation change. Precisely, the conformers
introduced in the calculation of the Raman intensities (Fig. 8B-D) were: extended chain conformers with $tg^-$ and $tg^+$ side chain orientations (B3LYP functional, Fig. 3C); helical conformers with $g^+g^+$ and $g^+g^-$ side chain orientations (M062X functional, Fig. 4B); extended chain conformers with $g^+g^+$ and $g^+g^-$ side chain orientations (ωB97XD functional, Fig. 3B). It can be seen that the Raman spectrum displayed in Fig. 8B (corresponding to B3LYP) provides the best agreement with the observed one (Fig. 8A), as far as the relative intensity of the $Y_5-Y_6$ doublet ($\rho$>1), and the distribution of calculated intensities in the whole middle wavenumber spectral region, are concerned. It should also be emphasized that the Raman spectra calculated by means of M062X and ωB97XD provide a reverse intensity ratio for the Tyr doublet ($\rho$<1) (Fig. 8C&D).

Assignments of the observed vibrational modes were displayed in Table 4 on the basis of the vibrational data relative to the lowest energy conformers issued from the DFT calculations by means of B3LYP functional (see Section 2.5 for details).

Considering these facts, the cartesian coordinates of the twenty four optimized conformers obtained at the B3LYP/6-311++G(d,p) (Table1) were provided as Supporting Information for further use of readers.

### 3.7. Can the Tyr doublet relative intensity be considered as a side chain orientation marker?

Our aim was to see whether the DFT calculated data collected from a relatively simple model compound such as the tripeptide Gly-Tyr-Gly, can help us understand the reasons behind the reversal of the $Y_5-Y_6$ doublet intensity ratio ($\rho$) (see Section 1 for details). For this, we consider those data obtained using B3LYP functional (see Sections 3.4, 3.5 and 3.6 for details). To calculate the Raman spectra, we followed a similar procedure as that detailed in Section 3.6. For each backbone conformation and a given $\chi_1$ ($g^-, g^+$ or t) orientation, the thermal average of the Raman spectra corresponding to its two lowest energy conformers
related one to another by the $\chi_2$ ($g^- \leftrightarrow g^+$) orientation change (Table 1), was computed. More precisely, extended chain conformers with $g^-$ and $g^+$ side chain orientations (Fig. 3C), helical conformers with $g^-g^+$ and $g^+g^-$ side chain orientations (Fig. 4A), pP-II conformers with $g^-g^+$ and $g^+g^-$ side chain orientations (Fig. 5A), and classic $\gamma$-turn with $g^-g^+$ and $g^+g^+$ side chain orientations (Fig. 6A), were introduced in thermal average calculations. Naturally, each obtained spectrum represents somehow a backbone conformation and one of the three possible ($g^-, g^+$ or $t$) orientations of the side chain $\chi_1$ torsion angle. In Fig. 9 the twelve calculated spectra are shown as the elements of a 4x3 table. While each row corresponds to one of the four backbone conformations (extended chain, helix, pP-II and classic $\gamma$-turn), each column represents one of the possible $\chi_1$ ($g^-, g^+$ and $t$) orientations.

These spectra lead us to an important conclusion: whatever the selected backbone conformation, the $\chi_1$ orientation plays a crucial role in the $\rho$ value reversal. While $g^-$ and $t$ orientations of $\chi_1$ leads to a doublet with $\rho>1$, its $g^+$ orientation provides a spectrum with $\rho<1$.

### 4. Concluding Remarks

In this report, attempt was made to bring clarification on the reversal of the Tyr characteristic Raman doublet intensity ratio ($\rho$). Room temperature Raman spectra of the tripeptide Gly-Tyr-Gly confirm the presence of the seven characteristic markers arising from the phenol ring involved in Tyr residue. The quite wavenumber constancy of these Raman lines upon selective deuteration (on labile hydrogen atoms) proves that they do not originate from the phenol hydroxyl vibrational motions. From the theoretical point of view, DFT calculations were carried out using three hybrid functionals (B3LYP, M062X and $\omega$B97XD) upon analysing the most occurrent ordered secondary structures in tripeptides ($\beta$-strand, helix, pP-II and $\gamma$-turn), and the privileged orientations of ($\chi_1, \chi_2$) torsion angles. The obtained data clearly showed that the energy landscape of the tripeptide is functional dependent. Particularly
the nature of the lowest energy conformer (ΔE=0) varies from one functional to another, i.e.,
an extended chain having a tg- side chain orientation, a helix with a g⁺g⁺ side chain
orientation, and an extended chain with a g⁺g⁺ side chain orientation, using B3LYP, M062X
and ωB97XD functional, respectively. It has also been evidenced that the possible χ₂ (g↔g⁺) orientation change (also characterized by the left↔right orientation change of O-H in phenol ring) is accompanied by a ΔE change comparable to thermal energy at room temperature (~0.6 kcal/mol), rendering possible the coexistence of both χ₂ (g⁺ and g⁻) orientations in normal conditions. Based on this fact, the calculated Raman spectrum to be compared with the observed one, was constructed by the thermal average of those related to the lowest energy conformer corresponding to ΔE=0, and its sister conformer obtained upon a χ₂ (g↔g⁺) conversion. The best agreement between the observed and calculated Raman spectrum was obtained with the data issued from the calculations using B3LYP functional. This strategy was then applied to each backbone secondary structure by constructing the thermal average of the calculated Raman spectra corresponding to its lowest energy conformer and the energetically sister conformer. The obtained spectra displayed in the Tyr doublet spectral region, highlighted that whatever the backbone conformation, the ρ value depends on the three possible orientations (g-, g⁺ and t) of the side chain χ₁ torsion angle, i.e., ρ>1 when χ₁ adopts g- or t orientations, and ρ>1 when χ₁ takes a g⁺ orientation. Beyond the fact that these theoretical results confirm our hypothesis concerning the relationship between ρ reversal and conformation, they pointed out the crucial importance of the side chain χ₁ torsion angle. In fact, χ₁ determines the location of the aromatic (phenol) ring with respect to the peptide backbone. With χ₁ (g-) and χ₁ (t) orientations, the phenol ring is stacked with a peptide bond (-CO-NH-) located at the N'ter and C'ter side of the Tyr residue, respectively, thus favouring the so-called π-π interactions between the two molecular segments. In contrast,
with $\chi_1$ ($g^+$) orientation the phenol ring is stacked with the N-H and C=O bonds belonging to the Tyr residue, and located at either side of its $C_\alpha$ atom (Figs. 3-6). One can also understand why the theoretical Raman spectrum in Gly-Tyr-Gly issued from the calculations using B3LYP shows a better agreement with the observed one. In fact, the two lowest energy conformers obtained by these calculations correspond both to a $\chi_1(t)$ orientation, which provides a $\rho$ value >1, reproducing well the spectral shape of the Tyr doublet observed in H₂O samples of the tripeptide (i.e., $\rho \sim 2$).

It is a matter of fact that the conclusions derived from the earliest works based on the relationship between the $\rho$ value reversal and H-bonding on phenol hydroxyl are to be definitively reconsidered, because the theoretical calculations described in the present work were performed without any explicit water molecule. However, it should also be reminded that the interaction with surrounding solvent molecules may perturb the conformational equilibrium between the conformers, inducing a more or less important perturbation in their energy landscape [48]. This can in turn introduce possible changes in the thermal average of the Raman spectra arising from the low energy conformers, leading to the modification of the observed Raman intensities. Considering this view, we can suggest an explanation for the difference observed in $\rho$ value in going from H₂O ($\rho \sim 2$) to D₂O ($\rho \sim 1$) samples of Gly-Tyr-Gly, based on the possible equilibrium change between conformers having $\chi_1(t)$ and $\chi_1(g^+)$ orientations, reinforced by the H-D exchange in phenol hydroxyl group.

## Acknowledgements

The theoretical calculations described here were granted access to the HPC resources of IDRIS under the allocation 2023-AD010805065R2 made by Grand Equipment National de Calcul Intensif (GENCI). The authors would like to thank Dr. Fernando Pflüger for his suggestions on the structural analysis of tripeptides.
References

[1] R. C. Lord, N. T. Yu, Laser-excited Raman spectroscopy of biomolecules: I. Native lysozyme and its constituent amino acids, J. Mol. Biol. 50 (1970) 509-524.

[2] M. N. Siamwiza, R. C. Lord, M. C. Chen, T. Takamatsu, I. Harada, H. Matsuura, T. Shimanouchi, Interpretation of the doublet at 850 and 830 cm⁻¹ in the Raman spectra of tyrosyl residues in proteins and certain model compounds, Biochemistry 14 (1975), 4870-4876.

[3] P. G. Hildebrandt, R. A. Copeland, T. G. Spiro, J. Otlewski, M. Laskowski Jr and F. G. Prendergast, Tyrosine hydrogen-bonding and environmental effects in proteins probed by ultraviolet resonance Raman spectroscopy, Biochemistry 27 (1988) 5426-5433.

[4] M. M. Whittaker, V. L. DeVito, S. A. Asher, J. W. Whittaker, Resonance Raman evidence for tyrosine involvement in the radical site of galactose oxidase, J. Biol. Chem. 264 (1989) 7104-7106.

[5] S. A. Asher, P. J. Larkin, J. Teraoka, Ultraviolet resonance Raman and absorption difference spectroscopy of myoglobins: titration behavior of individual tyrosine residues, Biochemistry 30 (1991) 5944-5954.

[6] S. A. Overman, K. L. Aubrey, N. S. Vispo, G. Cesareni, G. J. Thomas Jr, Novel tyrosine markers in Raman spectra of wild-type and mutant (Y21M and Y24M) Ff virions indicate unusual environments for coat protein phenoxyls, 33 (1994) 1037-1042.

[7] Z. Q. Wen, G. J. Thomas Jr., UV resonance Raman spectroscopy of DNA and protein constituents of viruses: assignments and cross sections for excitations at 257, 244, 238, and 229 nm, Biopolymers 45 (1998) 247-256.

[8] G. J. Thomas Jr, Raman spectroscopy of protein and nucleic acid assemblies, Annu. Rev. Biophys. Biomol. Struct. 28 (1999) 1-27.

[9] Z. Arp, D. Autrey, J. Laane, S. A. Overman, G. J. Thomas Jr, Tyrosine Raman signatures of the filamentous virus Ff are diagnostic of non-hydrogen-bonded phenoxyls: demonstration by Raman and infrared spectroscopy of p-cresol vapor, Biochemistry 40 (2001), 2522-2529.

[10] J. M. Benevides, S. A. Overman, G. J. Thomas Jr, in Raman Spectroscopy of Proteins, in Current Protocols in Protein Science (Eds: J. E. Coligan, B. M. Dunn, H. L. Ploegh, D. W. Speicher, P. T. Wingfield), John Wiley & Sons Inc, New York, 2003, 17.8.1-17.8.35.

[11] A. Pandit, N. Fay, L. Bordes, C. Valéry, R. Cherif-Cheikh, B. Robert, F. Artzner, M. Paternostre, Self-assembly of the octapeptide lanreotide and lanreotide-based derivatives: the role of the aromatic residues, J. Pept. Sci. 14 (2008) 66-75.

[12] F. Wei, D. Zhang D, N. J. Halas, J. D. J. Hartgerink, Aromatic amino acids providing characteristic motifs in the Raman and SERS spectroscopy of peptides, Phys. Chem. B 112 (2008) 9158-9164.
[13] B. Hernández, F. Pflüger, A. Adenier, S. G. Kruglik, M. Ghomi, Vibrational analysis of amino acids and short peptides in hydrated media. VIII. Amino acids with aromatic side chains: L-phenylalanine, L-tyrosine, and L-tryptophan, J. Phys. Chem. B 114 (2010) 15319-15330.

[14] F. Pflüger, B. Hernández, M. Ghomi, Vibrational analysis of amino acids and short peptides in hydrated media. VII. Energy landscapes, energetic and geometrical features of L-histidine with protonated and neutral side chains, J. Phys. Chem. B 114 (2010) 9072-9083.

[15] H. Takeuchi, UV Raman markers for structural analysis of aromatic side chains in proteins, Anal. Sci. 27 (2011) 1077-1086.

[16] B. Hernández, F. Pflüger, S. G. Kruglik, M. Ghomi, Characteristic Raman lines of phenylalanine analyzed by a multiconformational approach, J. Raman Spectrosc. 44 (2013) 827-833.

[17] B. Sjöberg, S. Foley, B. Cardey, M. Enescu, An experimental and theoretical study of the amino acid side chain Raman bands in proteins, Spectrochim. Acta A 128 (2014) 300-311.

[18] B. Hernández, Y. M. Coïc, F. Pflüger, S. G. Kruglik, M. Ghomi, All characteristic Raman markers of tyrosine and tyrosinate originate from phenol ring fundamental vibrations, J. Raman Spectrosc. 47 (2016) 210-220.

[19] G. E. Schulz, R. H. Schirmer, Principles of protein structure, Berlin: Springer-Verlag, 1979.

[20] J. R. Avilés Moreno, M. M. Quesada Moreno, F. Partal Ureña, J. J. López González, Conformational preference of short aromatic amino acids from the FT-IR, FT-Raman and Far-IR spectroscopies, and quantum chemical calculations: L-phenylalanine and L-tyrosine, Tetrahedron, Asymmetry 23 (2012) 1084-1092.

[21] J. B. Cabalo, S. K. Saikin, E. D. Emmons, D. Rappoport, A. Aspuru-Guzik, State-by-State Investigation of Destructive Interference in Resonance Raman Spectra of Neutral Tyrosine and the Tyrosinate Anion with the Simplified Sum-over-States Approach, J. Phys. Chem. A 118 (2014) 9675-9686.

[22] B. Hernández, S. Sanchez-Cortes, M. Ghomi, Energetic, conformational and vibrational features of the tripeptide (Gly)₃. Data from MP2 and DFT calculations, Comput. Theor. Chem. 1220 (2023) 113989.

[23] R. Schweitzer-Stenner, F. Eker, Q. Huang, K. Griebenow, Dihedral Angles of Trialanine in D₂O Determined by Combining FTIR and Polarized Visible Raman Spectroscopy, J. Am. Chem. Soc. 123 (2001) 9628-9633.

[24] R. Schweitzer-Stenner, Dihedral Angles of Tripeptides in Solution Directly Determined by Polarized Raman and FTIR Spectroscopy, Biophys. J. 83 (2002) 523-532.

[25] A. Hagarman, T. J. Measey, D. Mathieu, H. Schwalbe, R. Schweitzer-Stenner, Intrinsic Propensities of Amino Acid Residues in GxG Peptides Inferred from Amide I' Band Profiles and NMR Scalar Coupling Constants, J. Am. Chem. Soc. 132 (2010) 540-551.
[26] R Schweitzer-Stenner, A. Hagarman, S. Toal, D. Mathieu, H. Schwalbe, Disorder and order in unfolded and disordered peptides and proteins: A view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains, Proteins 81 (2013) 955-967.

[27] B. Hernández, F. Pflüger, S.G. Kruglik, M. Ghomi, Multiconformational analysis of tripeptides upon consideration of implicit and explicit hydration effects, J. Mol. Graph. Modell. 102 (2021) 107790.

[28] W.C. Chan, P.D. White Fmoc Solid Phase Peptide Synthesis. A Practical Approach. Oxford: Oxford University Press, 2000.

[29] R. Kishore, P. Balaram, Stabilization of γ-turn conformations in peptides by disulfide bridging, Biopolymers 24 (1985) 2041-2043.

[30] E. J. Milner-White, B. M. Ross, R. Ismail, K. Belhadj-Mostefa, R. Poet, One type of gamma-turn, rather than the other gives rise to chain-reversal in proteins, J. Mol. Biol. 204 (1988) 777-782.

[31] N. Mardirossian, M. Head-Gordon, Thirty years of density functional theory in computational chemistry: an overview and extensive assessment of 200 density functionals, Mol. Phys. 115 (2017) 2315-2372.

[32] W. Kohn, L. J. Sham, Self-consistent equations including exchange and correlation effects, Phys. Rev. 140 (1965) A1133-A1138.

[33] C. Møller, M. S. Plesset, Note on an Approximation Treatment for Many-Electron Systems, Phys. Rev. 46 (1934) 618-622.

[34] T. Benighaus, R. A. DiStasio, Jr., R. C. Lochan, J. D. Chai, M. Head-Gordon, Semiempirical Double-Hybrid Density Functional with Improved Description of Long-Range Correlation, J. Phys. Chem. A 112 (2008) 2702-2712.

[35] J. D. Chai, M. Head-Gordon, Systematic optimization of long-range corrected hybrid density functionals, J. Chem. Phys. 128 (2008) 084106.

[36] J. D. Chai, M. Head-Gordon, Long-range corrected hybrid density functionals with damped atom-atom dispersion corrections, Phys. Chem. Chem. Phys. 10 (2008) 6615-6620.

[37] Y. Zhao, D.G. Truhlar, The M06 suite of density functional for main group thermochemistry, thermochemical kinetics, noncovalent interactions, excited states, and transition elements: two new functionals and systematic testing of four M06-class functionals and 12 other functionals, Theor. Chem. Acc. 120 (2008) 215-241.

[38] C. Lee, W. Yang, R. G. Parr, Development of the Colle-Salvetti correlation-energy formula into a functional of the electron density, Phys. Rev. B: Condens. Matter Mater. Phys. 37 (1988) 785-789.

[39] A. D. Becke, Density-functional thermochemistry. III. The role of exact exchange, J. Chem. Phys. 98 (1993) 5648-5652.
[40] V. Barone, M. Cossi, Quantum calculation of molecular energies and energy gradients in solution by a conductor solvent model, J. Phys. Chem. A 102 (1988) 1995-2001.

[41] M. Cossi, N. Rega, G. Scalmani and V. Barone, Energies, structures, and electronic properties of molecules in solution with the C-PCM solvation model, J. Comput. Chem. 24 (2003) 669-681.

[42] M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria, M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone, G. A. Petersson, H. Nakatsuji, X. Li, M. Caricato, A. V. Marenich, J. Bloino, B. G. Janesko, R. Gomperts, B. Mennucci, H. P. Hratchian, J. V. Ortiz, A. F. Izmaylov, J. L. Sonnenberg, D. Williams-Young, F. Ding, F. Lipparini, F. Egidi, J. Goings, B. Peng, A. Petrone, T. Henderson, D. Ranasinghe, V. G. Zakrzewski, J. Gao, N. Rega, G. Zheng, W. Liang, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, K. Throssell, J. A. Montgomery, Jr., J. E. Peralta, F. Ogliaro, M. J. Bearpark, J. J. Heyd, E. N. Brothers, K. N. Kudin, V. N. Staroverov, T. A. Keith, R. Kobayashi, J. Normand, K. Raghavachari, A. P. Rendell, J. C. Burant, S. S. Iyengar, J. Tomasi, M. Cossi, J. M. Millam, M. Klene, C. Adamo, R. Cammi, J. W. Ochterski, R. L. Martin, K. Morokuma, O. Farkas, J. B. Foresman, and D. J. Fox, Gaussian 16, Revision C.01, Gaussian, Inc., Wallingford CT, 2019.

[43] G. Keresztury, Raman spectroscopy: theory, in: J.M. Chalmers, P.R. Griffiths (Eds.), Handbook of Vibrational Spectroscopy, vol. 1, Wiley & Sons, 2002, pp.71–87.

[44] S. Califano, Vibrational States, Wiley, New York, 1976.

[45] B. Hernández, F.-Z. Boukhalfa-Heniche, O. Seksek, Y. M. Coïc, M. Ghomi, Secondary Conformation of Short Lysine- and Leucine-Rich Peptides Assessed by Optical Spectroscopies: Effect of Chain Length, Concentration, Solvent, and Time, Biopolymers 81 (2006) 8–19.

[46] B. Hernández, F. Pflüger, A. Adenier, M. Nsangou, S. G. Kruglik, M. Ghomi, Energy maps, side chain conformational flexibility, and vibrational features of polar amino acids L–serine and L-threonine in aqueous environment, J. Chem. Phys. 135 (2011) 055101.

[47] B. Hernández, F. Pflüger, A. Adenier, S. G. Kruglik, M. Ghomi, Side chain flexibility and protonation states of sulfur atom containing amino acids, Phys. Chem. Chem. Phys. 13 (2011) 17284-17294.

[48] B. Hernández, F. Pflüger, M. Dauchez, M. Ghomi, Privileged hydration sites in aromatic side chains: effect on conformational equilibrium, Phys. Chem. Chem. Phys. 19 (2017) 28684-28695.
<table><caption>Table 1 Conformational angles and relative energies obtained by means of B3LYP functional on the cationic species of Gly-Tyr-Gly conformers embedded in a solvent continuum</caption><thead><tr><th rowspan="2">Conformers</th><th colspan="8">Backbone</th><th colspan="2">Side-chain</th><th rowspan="2">d<sub>HB</sub></th><th rowspan="2">ΔE</th></tr><tr><th>Ψ<sub>1</sub></th><th>ω<sub>1</sub></th><th>φ<sub>2</sub></th><th>Ψ<sub>2</sub></th><th>ω<sub>2</sub></th><th>φ<sub>3</sub></th><th>Ψ<sub>3</sub></th><th>χ<sub>1</sub></th><th>χ<sub>2</sub></th></tr></thead><tbody><tr><td>Extended chain/g<sup>-</sup>g<sup>-</sup></td><td>+175.5</td><td>+176.4</td><td>-127.9</td><td>+147.1</td><td>+175.7</td><td>-178.7</td><td>+178.8</td><td>-62.4</td><td>-81.1</td><td></td><td>+0.72</td></tr><tr><td>Extended chain/g<sup>-</sup>g<sup>+</sup></td><td>+175.6</td><td>+176.3</td><td>-127.9</td><td>+147.1</td><td>+175.7</td><td>-178.8</td><td>+178.9</td><td>-62.9</td><td>+98.7</td><td></td><td>+0.78</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>-</sup></td><td>+171.1</td><td>+174.3</td><td>-153.3</td><td>+165.1</td><td>+178.4</td><td>+179.3</td><td>+179.7</td><td>+61.5</td><td>-89.4</td><td></td><td>+0.12</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>+</sup></td><td>+167.3</td><td>+174.5</td><td>-154.1</td><td>+165.2</td><td>+178.1</td><td>-179.8</td><td>+179.3</td><td>+62.0</td><td>+91.5</td><td></td><td>+0.93</td></tr><tr><td>Extended chain/tg<sup>-</sup></td><td>+175.6</td><td>+176.4</td><td>-127.9</td><td>+147.1</td><td>+175.7</td><td>-178.7</td><td>+178.8</td><td>-62.4</td><td>-81.1</td><td></td><td>0.</td></tr><tr><td>Extended chain/tg<sup>+</sup></td><td>+170.1</td><td>+177.2</td><td>-150.1</td><td>+143.7</td><td>+176.6</td><td>+178.9</td><td>-179.3</td><td>-174.9</td><td>+78.7</td><td></td><td>0.04</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>-</sup></td><td>+170.9</td><td>+176.8</td><td>-80.3</td><td>+144.7</td><td>+174.0</td><td>-83.9</td><td>+173.5</td><td>-62.2</td><td>-71.5</td><td></td><td>+0.61</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>+</sup></td><td>+172.4</td><td>+175.2</td><td>-127.8</td><td>+145.4</td><td>+175.3</td><td>-83.0</td><td>+171.4</td><td>-63.3</td><td>+96.9</td><td></td><td>+0.70</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>-</sup></td><td>+167.3</td><td>+175.1</td><td>-86.1</td><td>+159.9</td><td>+177.0</td><td>-85.0</td><td>+170.6</td><td>+55.4</td><td>-97.7</td><td></td><td>+2.56</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>+</sup></td><td>+167.4</td><td>+174.9</td><td>-85.2</td><td>+160.6</td><td>+177.4</td><td>-83.3</td><td>+170.3</td><td>+55.3</td><td>+83.2</td><td></td><td>+2.46</td></tr><tr><td>pP-II/tg<sup>-</sup></td><td>+170.4</td><td>+174.1</td><td>-77.9</td><td>+165.6</td><td>+177.8</td><td>-75.7</td><td>+169.9</td><td>-160.2</td><td>-109.3</td><td></td><td>+2.94</td></tr><tr><td>pP-II/tg<sup>+</sup></td><td>+168.0</td><td>+174.4</td><td>-85.4</td><td>+169.5</td><td>+178.2</td><td>-79.5</td><td>+175.2</td><td>-160.1</td><td>+67.6</td><td></td><td>+3.37</td></tr><tr><td>Helix/g<sup>-</sup>g<sup>-</sup></td><td>-165.6</td><td>-167.8</td><td>-67.1</td><td>-24.2</td><td>+179.0</td><td>-88.3</td><td>+0.3</td><td>-61.5</td><td>-69.5</td><td>2.18</td><td>+1.79</td></tr><tr><td>Helix/g<sup>-</sup>g<sup>+</sup></td><td>-165.6</td><td>-167.4</td><td>-67.0</td><td>-24.5</td><td>+179.2</td><td>-88.0</td><td>+0.2</td><td>-60.6</td><td>+112.1</td><td>2.18</td><td>+2.05</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>-</sup></td><td>-163.9</td><td>-169.4</td><td>-74.8</td><td>-12.5</td><td>+174.8</td><td>-91.3</td><td>-0.2</td><td>+55.7</td><td>-99.5</td><td>2.28</td><td>+3.33</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>+</sup></td><td>-164.1</td><td>-169.1</td><td>-75.1</td><td>-12.4</td><td>+174.7</td><td>-91.3</td><td>-0.2</td><td>+56.2</td><td>+81.3</td><td>2.28</td><td>+3.50</td></tr><tr><td>Helix/tg<sup>-</sup></td><td>-163.8</td><td>-168.3</td><td>-61.4</td><td>-34.2</td><td>-178.7</td><td>-85.8</td><td>+0.6</td><td>-173.6</td><td>-105.3</td><td>2.15</td><td>+3.57</td></tr><tr><td>Helix/tg<sup>+</sup></td><td>-163.6</td><td>-168.1</td><td>-61.4</td><td>-34.3</td><td>-178.7</td><td>-86.0</td><td>+0.8</td><td>-173.3</td><td>+76.7</td><td>2.15</td><td>+3.36</td></tr><tr><td>Classic γ-turn/g<sup>-</sup>g<sup>-</sup></td><td>+168.1</td><td>+171.3</td><td>+76.3</td><td>-55.9</td><td>-177.7</td><td>+174.9</td><td>+179.2</td><td>-58.6</td><td>-72.3</td><td>2.06</td><td>+4.85</td></tr><tr><td>Classic γ-turn/g<sup>-</sup>g<sup>+</sup></td><td>+168.0</td><td>+171.2</td><td>+76.4</td><td>-55.9</td><td>-177.7</td><td>+174.8</td><td>+179.2</td><td>-58.7</td><td>+107.9</td><td>2.06</td><td>+4.63</td></tr><tr><td>Classic γ-turn/g<sup>+</sup>g<sup>-</sup></td><td>+163.7</td><td>+170.4</td><td>+57.6</td><td>-37.8</td><td>-179.6</td><td>-83.1</td><td>+169.9</td><td>+64.0</td><td>-98.6</td><td>1.83</td><td>+8.98</td></tr><tr><td>Classic γ-turn/g<sup>+</sup>g<sup>+</sup></td><td>+163.9</td><td>+170.1</td><td>+57.6</td><td>-38.1</td><td>-179.6</td><td>-83.4</td><td>+170.3</td><td>+64.5</td><td>+83.4</td><td>1.83</td><td>+8.83</td></tr><tr><td>Classic γ-turn/tg<sup>-</sup></td><td>+166.7</td><td>+172.6</td><td>+76.9</td><td>-64.0</td><td>-179.2</td><td>+174.4</td><td>-177.8</td><td>-170.6</td><td>-101.1</td><td>2.10</td><td>+5.17</td></tr><tr><td>Classic γ-turn/tg<sup>+</sup></td><td>+166.7</td><td>+172.6</td><td>+76.9</td><td>-63.9</td><td>-179.2</td><td>+174.6</td><td>-177.8</td><td>-170.9</td><td>+78.2</td><td>2.10</td><td>+5.14</td></tr></tbody></table>

Conformational angles are expressed in degrees (see Fig. 1A&B for their definition). Relative energies (ΔE) are reported in kcal/mol (see Sections 2.4 and 2.5 for details). Graphical representations of conformers are displayed in Fig.3-6. d<sub>HB</sub> represents the intramolecular hydrogen bond formed in helical (Fig. 4) and Classic γ-turn (Fig. 6) conformers.
<table><caption>Table 2 Conformational angles and relative energies obtained by means of M062X functional on the cationic species of Gly-Tyr-Gly conformers embedded in a solvent continuum</caption><thead><tr><th rowspan="2">Conformers</th><th colspan="6">Backbone</th><th colspan="2">Side-chain</th><th rowspan="2">d<sub>HB</sub></th><th rowspan="2">ΔE</th></tr><tr><th>ψ<sub>1</sub></th><th>ω<sub>1</sub></th><th>φ<sub>2</sub></th><th>ψ<sub>2</sub></th><th>ω<sub>2</sub></th><th>φ<sub>3</sub></th><th>ψ<sub>3</sub></th><th>χ<sub>1</sub></th><th>χ<sub>2</sub></th></tr></thead><tbody><tr><td>Helix/g<sup>-</sup>g<sup>-</sup></td><td>-164.7</td><td>-168.0</td><td>-63.9</td><td>-26.6</td><td>-179.2</td><td>-76.9</td><td>-7.0</td><td>-58.6</td><td>-69.9</td><td>2.07</td><td>+0.42</td></tr><tr><td>Helix/g<sup>-</sup>g<sup>+</sup></td><td>-165.2</td><td>-167.8</td><td>-64.1</td><td>-26.5</td><td>-179.2</td><td>-77.0</td><td>-7.0</td><td>-58.0</td><td>+110.6</td><td>2.07</td><td>+0.84</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>-</sup></td><td>-167.7</td><td>-170.3</td><td>-67.7</td><td>-19.2</td><td>+179.8</td><td>-77.8</td><td>-9.1</td><td>+61.7</td><td>-97.9</td><td>2.15</td><td>+0.51</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>+</sup></td><td>-167.7</td><td>-170.2</td><td>-67.5</td><td>-19.7</td><td>180.0</td><td>-78.8</td><td>-8.1</td><td>+61.5</td><td>+80.8</td><td>2.14</td><td>0.0</td></tr><tr><td>Helix/tg<sup>-</sup></td><td>-164.6</td><td>-170.3</td><td>-57.8</td><td>-38.3</td><td>-176.0</td><td>-78.3</td><td>-3.7</td><td>-177.8</td><td>-97.8</td><td>2.05</td><td>+1.42</td></tr><tr><td>Helix/tg<sup>+</sup></td><td>-164.6</td><td>-170.4</td><td>-57.9</td><td>-38.1</td><td>-176.1</td><td>-78.4</td><td>-3.5</td><td>-177.9</td><td>+80.4</td><td>2.05</td><td>+1.50</td></tr><tr><td>Extended chain/g<sup>-</sup>g<sup>-</sup></td><td>180.0</td><td>-176.6</td><td>-145.5</td><td>+155.1</td><td>+175.7</td><td>-179.3</td><td>+179.9</td><td>-58.7</td><td>-86.6</td><td></td><td>+1.43</td></tr><tr><td>Extended chain/g<sup>-</sup>g<sup>+</sup></td><td>+179.9</td><td>-176.7</td><td>-145.3</td><td>+154.9</td><td>+175.7</td><td>-179.4</td><td>180.0</td><td>-59.1</td><td>+90.1</td><td></td><td>+1.25</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>-</sup></td><td>+165.8</td><td>+177.5</td><td>-162.8</td><td>+172.7</td><td>+166.7</td><td>-148.0</td><td>-179.6</td><td>+69.2</td><td>-81.8</td><td></td><td>+0.79</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>+</sup></td><td>+165.9</td><td>+177.6</td><td>-162.8</td><td>+172.6</td><td>+166.8</td><td>-147.4</td><td>-179.5</td><td>+68.9</td><td>+95.4</td><td></td><td>+0.78</td></tr><tr><td>Extended chain/tg<sup>-</sup></td><td>+168.3</td><td>+178.4</td><td>-157.0</td><td>+147.3</td><td>+161.3</td><td>-156.2</td><td>+167.6</td><td>+173.7</td><td>-92.2</td><td></td><td>+0.60</td></tr><tr><td>Extended chain/tg<sup>+</sup></td><td>+168.4</td><td>+178.3</td><td>-156.8</td><td>+147.5</td><td>+161.6</td><td>-156.6</td><td>+169.1</td><td>+174.3</td><td>+82.6</td><td></td><td>+0.76</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>-</sup></td><td>+165.3</td><td>+172.9</td><td>-61.2</td><td>+150.4</td><td>+167.3</td><td>-65.9</td><td>+165.1</td><td>-58.8</td><td>-69.2</td><td></td><td>+1.89</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>+</sup></td><td>+165.3</td><td>+173.0</td><td>-61.1</td><td>+150.6</td><td>+167.0</td><td>-65.8</td><td>+165.2</td><td>-58.6</td><td>+110.9</td><td></td><td>+2.11</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>-</sup></td><td>+163.9</td><td>+170.8</td><td>-65.0</td><td>+156.4</td><td>+175.8</td><td>-72.8</td><td>+165.2</td><td>+52.1</td><td>-98.8</td><td></td><td>+1.99</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>+</sup></td><td>+164.0</td><td>+170.7</td><td>-64.8</td><td>+156.6</td><td>+175.9</td><td>-72.9</td><td>+165.3</td><td>+52.1</td><td>+79.9</td><td></td><td>+1.91</td></tr><tr><td>pP-II/tg<sup>-</sup></td><td>+164.7</td><td>+169.7</td><td>-65.0</td><td>+163.6</td><td>+173.0</td><td>-63.9</td><td>+165.9</td><td>-163.3</td><td>-106.4</td><td></td><td>+2.59</td></tr><tr><td>pP-II/tg<sup>+</sup></td><td>+164.6</td><td>+169.6</td><td>-64.8</td><td>+163.3</td><td>+173.6</td><td>-64.2</td><td>+166.3</td><td>-163.1</td><td>+74.9</td><td></td><td>+2.93</td></tr></tbody></table>

Conformational angles are expressed in degrees (see Fig. 1A&B for their definition). Relative energies (ΔE) are reported in kcal/mol (see Sections 2.4 and 2.5 for details). Graphical representations of conformers are displayed in Fig.3-6. d<sub>HB</sub> represents the intramolecular hydrogen bond in formed in helical conformers (Fig. 4).
<table><caption>Table 3 Conformational angles and relative energies obtained by means of ωB97XD functional on the cationic species of Gly-Tyr-Gly conformers embedded in a solvent continuum</caption><thead><tr><th rowspan="2">Conformers</th><th colspan="8">Backbone</th><th colspan="2">Side-chain</th><th rowspan="2">d<sub>HB</sub></th><th rowspan="2">ΔE</th><th rowspan="2">ΔE'</th></tr><tr><th>Ψ<sub>1</sub></th><th>ω<sub>1</sub></th><th>φ<sub>2</sub></th><th>Ψ<sub>2</sub></th><th>ω<sub>2</sub></th><th>φ<sub>3</sub></th><th>Ψ<sub>3</sub></th><th>χ<sub>1</sub></th><th>χ<sub>2</sub></th></tr></thead><tbody><tr><td>Extended chain/g<sup>-</sup>g<sup>-</sup></td><td>+176.6</td><td>-177.5</td><td>-141.2</td><td>+153.7</td><td>+175.5</td><td>-179.3</td><td>+179.2</td><td>-59.2</td><td>-87.7</td><td></td><td>+1.09</td><td>+0.89</td></tr><tr><td>Extended chain/g<sup>-</sup>g<sup>+</sup></td><td>+176.7</td><td>-177.5</td><td>-141.2</td><td>+153.7</td><td>+175.5</td><td>-179.3</td><td>+179.2</td><td>-59.7</td><td>+89.4</td><td></td><td>+1.23</td><td>+1.04</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>-</sup></td><td>+170.3</td><td>+178.0</td><td>-162.3</td><td>+172.7</td><td>+168.2</td><td>-145.8</td><td>+175.2</td><td>+68.3</td><td>-82.6</td><td></td><td>+0.83</td><td>+0.83</td></tr><tr><td>Extended chain/g<sup>+</sup>g<sup>+</sup></td><td>+170.3</td><td>+178.0</td><td>-162.3</td><td>+172.7</td><td>+168.2</td><td>-145.1</td><td>+175.7</td><td>+68.2</td><td>+95.2</td><td></td><td>0.0</td><td>0.0</td></tr><tr><td>Extended chain/tg<sup>-</sup></td><td>+167.7</td><td>+178.5</td><td>-156.3</td><td>+150.9</td><td>+169.1</td><td>-169.4</td><td>+175.9</td><td>-179.3</td><td>-99.9</td><td></td><td>+1.51</td><td>+1.50</td></tr><tr><td>Extended chain/tg<sup>+</sup></td><td>+167.6</td><td>+178.6</td><td>-156.4</td><td>+150.6</td><td>+168.8</td><td>-169.3</td><td>+175.7</td><td>-179.9</td><td>+77.6</td><td></td><td>+1.59</td><td>+1.58</td></tr><tr><td>Helix/g<sup>-</sup>g<sup>-</sup></td><td>-163.8</td><td>-167.6</td><td>-66.0</td><td>-25.5</td><td>+178.8</td><td>-82.6</td><td>-2.8</td><td>-56.8</td><td>-70.1</td><td>2.08</td><td>+1.08</td><td>+1.51</td></tr><tr><td>Helix/g<sup>-</sup>g<sup>+</sup></td><td>-164.1</td><td>-167.4</td><td>-66.1</td><td>-25.6</td><td>+178.9</td><td>-82.7</td><td>-2.7</td><td>-56.4</td><td>+109.4</td><td>2.09</td><td>+1.59</td><td>+2.03</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>-</sup></td><td>-166.1</td><td>-170.1</td><td>-68.2</td><td>-19.9</td><td>+177.5</td><td>-85.5</td><td>-1.8</td><td>+59.2</td><td>-97.7</td><td>2.12</td><td>+1.63</td><td>+2.16</td></tr><tr><td>Helix/g<sup>+</sup>g<sup>+</sup></td><td>-166.3</td><td>-170.6</td><td>-68.3</td><td>-19.9</td><td>+177.6</td><td>-85.5</td><td>-1.9</td><td>+59.4</td><td>+80.9</td><td>2.13</td><td>+1.83</td><td>+2.37</td></tr><tr><td>Helix/tg<sup>-</sup></td><td>-165.1</td><td>-170.7</td><td>-58.5</td><td>-37.7</td><td>-177.9</td><td>-81.4</td><td>-1.0</td><td>-176.6</td><td>-100.9</td><td>2.07</td><td>+2.29</td><td>+2.73</td></tr><tr><td>Helix/tg<sup>+</sup></td><td>-165.0</td><td>-170.7</td><td>-58.6</td><td>-37.6</td><td>-177.9</td><td>-81.6</td><td>-1.0</td><td>-176.4</td><td>+78.1</td><td>2.07</td><td>+2.48</td><td>+2.91</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>-</sup></td><td>+166.5</td><td>+176.8</td><td>-73.8</td><td>+149.5</td><td>+169.3</td><td>-77.9</td><td>+174.9</td><td>-58.8</td><td>-69.2</td><td></td><td>+3.04</td><td>+2.99</td></tr><tr><td>pP-II/g<sup>-</sup>g<sup>+</sup></td><td>+166.2</td><td>+176.3</td><td>-72.6</td><td>+148.7</td><td>+169.0</td><td>-79.1</td><td>+175.7</td><td>-62.3</td><td>+105.5</td><td></td><td>+2.83</td><td>+2.76</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>-</sup></td><td>+170.3</td><td>+173.1</td><td>-75.6</td><td>+161.7</td><td>+177.5</td><td>-84.6</td><td>+167.0</td><td>+56.8</td><td>-95.9</td><td></td><td>+2.48</td><td>+2.70</td></tr><tr><td>pP-II/g<sup>+</sup>g<sup>+</sup></td><td>+170.0</td><td>+173.1</td><td>-75.7</td><td>+161.8</td><td>+177.5</td><td>-84.5</td><td>+167.1</td><td>+56.7</td><td>+82.7</td><td></td><td>+2.50</td><td>+2.73</td></tr><tr><td>pP-II/tg<sup>-</sup></td><td>+167.4</td><td>+172.4</td><td>-72.8</td><td>+164.9</td><td>+175.3</td><td>-74.7</td><td>+177.4</td><td>-163.0</td><td>-107.3</td><td></td><td>+3.81</td><td>+4.03</td></tr><tr><td>pP-II/tg<sup>+</sup></td><td>+167.9</td><td>+172.6</td><td>-73.0</td><td>+165.3</td><td>+174.8</td><td>-74.8</td><td>+177.5</td><td>-162.2</td><td>+72.4</td><td></td><td>+3.91</td><td>+4.13</td></tr><tr><td>Classic γ-turn/g<sup>-</sup>g<sup>-</sup></td><td>+165.5</td><td>+167.2</td><td>+79.1</td><td>-54.2</td><td>-178.0</td><td>+179.7</td><td>+178.2</td><td>-54.1</td><td>-74.9</td><td>2.03</td><td>+5.30</td><td>+5.47</td></tr><tr><td>Classic γ-turn/g<sup>-</sup>g<sup>+</sup></td><td>+165.4</td><td>+167.3</td><td>+79.0</td><td>-54.3</td><td>-178.0</td><td>+179.7</td><td>+178.2</td><td>-54.3</td><td>+102.6</td><td>2.03</td><td>+5.30</td><td>+5.49</td></tr><tr><td>Classic γ-turn/g<sup>+</sup>g<sup>-</sup></td><td>+164.1</td><td>+166.8</td><td>+57.8</td><td>-39.5</td><td>-177.6</td><td>-78.0</td><td>+167.1</td><td>+61.0</td><td>-96.5</td><td>1.80</td><td>+8.01</td><td>+8.46</td></tr><tr><td>Classic γ-turn/g<sup>+</sup>g<sup>+</sup></td><td>+164.1</td><td>+166.7</td><td>+57.7</td><td>-39.4</td><td>-177.6</td><td>-78.0</td><td>+167.1</td><td>+61.0</td><td>+82.4</td><td>1.80</td><td>+7.92</td><td>+8.27</td></tr><tr><td>Classic γ-turn/tg<sup>-</sup></td><td>+168.0</td><td>+173.9</td><td>+75.6</td><td>-66.0</td><td>-174.6</td><td>+166.8</td><td>-177.4</td><td>-177.0</td><td>-97.8</td><td>2.05</td><td>+5.38</td><td>+5.64</td></tr><tr><td>Classic γ-turn/tg<sup>+</sup></td><td>+168.0</td><td>+174.0</td><td>+75.5</td><td>-66.0</td><td>-174.6</td><td>+166.9</td><td>-177.5</td><td>-177.1</td><td>+79.9</td><td>2.05</td><td>+5.26</td><td>+5.52</td></tr></tbody></table>

Conformational angles are expressed in degrees (see Fig. 1A&B for their definition). Relative energies (ΔE and ΔE') are reported in kcal/mol (see Sections 2.4 and 2.5 for details). Graphical representations of conformers are displayed in Fig.3-6. d<sub>HB</sub> represents the intramolecular hydrogen bond formed in helical (Fig. 4) and classic γ-turn (Fig. 6) conformers.
<table><caption>Table 4 Observed and scaled theoretical wavenumbers (cm⁻¹) for the cationic species of Gly-Tyr-Gly</caption><thead><tr><th></th><th>Observed Raman (H<sub>2</sub>O)</th><th>Calculated B3LYP</th><th>Assignments</th></tr></thead><tbody><tr><td>amide I</td><td>1693 (m)</td><td>1689 (m)</td><td>v(C=O) (Gly<sup>3</sup>); v(C=O) (Gly<sup>1</sup>); v(C=O) (Gly<sup>2</sup>)</td></tr><tr><td>Y<sub>1</sub></td><td>1616 (s)</td><td>1624 (s)</td><td>v(C<sub>δ1</sub>-C<sub>ε1</sub>) (Tyr<sup>2</sup>); v(C<sub>δ2</sub>-C<sub>ε2</sub>) (Tyr<sup>2</sup>); v(C<sub>γ</sub>-C<sub>δ1</sub>) (Tyr<sup>2</sup>); v(C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td>Y<sub>2</sub></td><td>1600 (s,sh)</td><td>1606 (w)</td><td>v(C<sub>ε1</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); v(C<sub>γ</sub>-C<sub>δ2</sub>) (Tyr<sup>2</sup>); v(C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); v(C<sub>γ</sub>-C<sub>δ1</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td></td><td>1447 (m)</td><td>1477 (m)</td><td>C<sub>α</sub>-H<sub>2</sub> bend. (Gly<sup>3</sup>); C<sub>α</sub>-H<sub>2</sub> rock. (Gly<sup>3</sup>)</td></tr><tr><td></td><td>1434 (m)</td><td>1462 (m)</td><td>C<sub>β</sub>-H<sub>2</sub> bend. (Tyr<sup>2</sup>)</td></tr><tr><td></td><td>1403 (w)</td><td>1407 (w)</td><td>C<sub>α</sub>-H<sub>2</sub> rock. (Gly<sup>3</sup>); v(C-N<sub>amide</sub>) (Gly<sup>3</sup>); C<sub>α</sub>-H<sub>2</sub> bend. (Gly<sup>3</sup>); (C<sub>α</sub>-C<sub>amide</sub>) (Gly<sup>3</sup>)</td></tr><tr><td></td><td>1337 (m)</td><td>1357 (m)</td><td>C<sub>α</sub>-H<sub>2</sub> rock. (Gly<sup>1</sup>); C<sub>β</sub>-H<sub>2</sub> rock. (Tyr<sup>2</sup>); δ(N-C<sub>α</sub>-H) (Gly<sup>2</sup>)</td></tr><tr><td></td><td>1306 (w)</td><td>1309 (m)</td><td>C<sub>β</sub>-H<sub>2</sub> rock. (Tyr<sup>2</sup>); δ(N-C<sub>α</sub>-H) (Gly<sup>2</sup>)</td></tr><tr><td></td><td>1268 (s,br)</td><td>1250 (s)</td><td>v(C<sub>ζ</sub>-O<sub>η</sub>) (Tyr<sup>2</sup>); v(C<sub>δ2</sub>-C<sub>ε2</sub>) (Tyr<sup>2</sup>); v(C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); v(C<sub>δ1</sub>-C<sub>ε1</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td>Y<sub>3</sub></td><td>1209 (s)</td><td>1216 (s)</td><td>v(C<sub>β</sub>-C<sub>γ</sub>) (Tyr<sup>2</sup>); C<sub>β</sub>-H<sub>2</sub> rock. (Tyr<sup>2</sup>); v(C<sub>δ1</sub>-C<sub>ε1</sub>) (Tyr<sup>2</sup>); v(C<sub>δ2</sub>-C<sub>ε2</sub>) (Tyr<sup>2</sup>); v(C<sub>γ</sub>-C<sub>δ2</sub>) (Tyr<sup>2</sup>); δ(C<sub>δ1</sub>-C<sub>γ</sub>-C<sub>δ2</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td>Y<sub>4</sub></td><td>1178 (s)</td><td>1178 (s)</td><td>δ(C<sub>γ</sub>-C<sub>δ1</sub>-H) (Tyr<sup>2</sup>); δ(C<sub>ε1</sub>-C<sub>δ1</sub>-H) (Tyr<sup>2</sup>); δ(C<sub>ζ</sub>-O<sub>η</sub>-H) (Tyr<sup>2</sup>); δ(C<sub>ζ</sub>-C<sub>ε2</sub>-H) (Tyr<sup>2</sup>); C<sub>β</sub>-H<sub>2</sub> twist. (Tyr<sup>2</sup>); δ(C<sub>δ2</sub>-C<sub>ε2</sub>-H) (Tyr<sup>2</sup>); δ(C<sub>δ1</sub>-C<sub>ε1</sub>-H) (Tyr<sup>2</sup>); v(C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td></td><td>1106 (m,br)</td><td>1107 (w)</td><td>NH<sub>3</sub><sup>+</sup> asym. rock.(Gly<sup>1</sup>)); C<sub>α</sub>-H<sub>2</sub> twist. (Gly<sup>1</sup>); C<sub>α</sub>-H<sub>2</sub> wagg. (Gly<sup>1</sup>); δ(C<sub>α</sub>-C=O) (Gly<sup>1</sup>)</td></tr><tr><td></td><td>1025 (m)</td><td>1022 (m)</td><td>v(C<sub>α</sub>-C<sub>β</sub>) (Tyr<sup>2</sup>); v(C<sub>α</sub>-C) (Tyr<sup>2</sup>); δ(N-C<sub>α</sub>-C) (Tyr<sup>2</sup>); δ(C-C<sub>α</sub>-N) (Tyr<sup>2</sup>)</td></tr><tr><td></td><td>930 (m)</td><td>926 (w)</td><td>δ(N-C=O) (Gly<sup>1</sup>,Tyr<sup>2</sup>); v(C<sub>α</sub>-C) (Gly<sup>1</sup>); v(C<sub>α</sub>-N) (Gly<sup>1</sup>); δ(N-C=O) (Tyr<sup>2</sup>,Gly<sup>3</sup>); δ(C-N-C<sub>α</sub>) (Tyr<sup>2</sup>,Gly<sup>3</sup>)</td></tr><tr><td></td><td>875 (m,sh)</td><td>907 (s)</td><td>C<sub>β</sub>-H<sub>2</sub> wagg. (Tyr<sup>2</sup>); v(C<sub>α</sub>-C<sub>β</sub>) (Tyr<sup>2</sup>); v(C<sub>α</sub>-C) (Gly<sup>1</sup>)</td></tr><tr><td>Y<sub>5</sub></td><td>851 (s)</td><td>850 (s)</td><td>v(C<sub>γ</sub>-C<sub>δ1</sub>) (Tyr<sup>2</sup>); v(C<sub>β</sub>-C<sub>γ</sub>) (Tyr<sup>2</sup>); v(C<sub>ε1</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); v(C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); v(C<sub>γ</sub>-C<sub>δ1</sub>) (Tyr<sup>2</sup>); v(C<sub>ζ</sub>-O<sub>η</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td>Y<sub>6</sub></td><td>828 (m,sh)</td><td>834 (m)</td><td>ω(C<sub>ε2</sub>-H) (Tyr<sup>2</sup>); v(O<sub>η</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); ω(C<sub>δ2</sub>-H) (Tyr<sup>2</sup>); v(C<sub>α</sub>-C<sub>amide</sub>) (Gly<sup>3</sup>); ω(C<sub>δ1</sub>-H) (Tyr<sup>2</sup>)</td></tr><tr><td></td><td>805 (m,sh)</td><td>815 (w)</td><td>ω(C<sub>ε1</sub>-H) (Tyr<sup>2</sup>); ω(C<sub>δ1</sub>-H) (Tyr<sup>2</sup>); ω(C<sub>ε2</sub>-H) (Tyr<sup>2</sup>); τ(C<sub>γ</sub>-C<sub>δ1</sub>) (Tyr<sup>2</sup>); τ(C<sub>ε1</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>)</td></tr><tr><td>Y<sub>7</sub></td><td>643 (m)</td><td>647 (m)</td><td>δ(C<sub>γ</sub>-C<sub>δ1</sub>-C<sub>ε1</sub>) (Tyr<sup>2</sup>); δ(C<sub>γ</sub>-C<sub>δ2</sub>-C<sub>ε2</sub>) (Tyr<sup>2</sup>); δ(C<sub>δ1</sub>-C<sub>ε1</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>); δ(C<sub>δ2</sub>-C<sub>ε2</sub>-C<sub>ζ</sub>) (Tyr<sup>2</sup>)</td></tr></tbody></table>

In the first column the notations used for characteristic Raman lines are reported.

Observed: positions of the main peaks (cm⁻¹) taken from the room-temperature aqueous solution Raman spectrum from the cationic species of Gly-Tyr-Gly (20 mM, pH7) (Fig. 2C).

Calculated: scaled theoretical wavenumbers (cm⁻¹) obtained from the DFT calculations by means of B3LYP functional and 6-311++G(d,p) basis sets. See Fig. 8C and Section 3.6 for details concerning the calculation of Raman spectra. (s) strong, (m) middle, (w) weak, (sh) shoulder, br (broad).

Assignments: based on the potential energy distribution (PED) matrix derived from the vibrational calculations. v, δ, ω and τ designate bond-stretch, angular bending, out-of-plane bending and torsion internal coordinates. Bend., scissor., wagg. and rock. reflect the symmetrical coordinates associated to the CH₂ groups. Asym. rock. is relative to the terminal NH₃⁺ moiety. In parentheses, the residue (Gly¹, Tyr², Gly³), to which belongs the internal/symmetrical coordinate, is reported in parentheses. See also Fig. 1A&B for atomic nomenclature of backbone and side chain.
Figure captions

**Fig. 1** Structural parameters of the cationic species of the tripeptide Gly-Tyr-Gly. (A) Backbone torsion angles defined along the chemical bonds in going from N'ter (left) to C'ter (right) terminal groups. (B) torsion angles defined along the side chain chemical bonds. $\chi_2(g^+)$ and $\chi_2(g^-)$ orientations are presented in (A) and (B), respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

**Fig. 2** Raman spectra recorded in the 1800-600 $cm^{-1}$ spectral region. (A) Raw Raman spectrum obtained from the samples of Gly-Tyr-Gly dissolved in $H_2O$ (20 mM, pH 6.5). Red asterisk indicates the position of the most intense band assigned to TFA. (B) Buffer ($H_2O$) spectrum. (C) Buffer ($H_2O$) and TFA subtracted spectrum of the tripeptide Gly-Tyr-Gly; (D) Buffer ($D_2O$) and TFA subtracted spectrum of the tripeptide Gly-Tyr-Gly. The frame drawn with broken line delimit the spectral region in which the Tyr characteristic doublet and its satellite bands are observed.

**Fig. 3** Calculated Raman spectra displayed in the 900-800 $cm^{-1}$ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having an extended chain backbone. (A) conformers with $g^-$ and $g^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with $g^+$ and $g^-$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with $tg^-$ and $tg^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

**Fig. 4** Calculated Raman spectra displayed in the 900-800 $cm^{-1}$ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having a helical backbone. (A) conformers with $g^-$ and $g^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with $g^+$ and $g^-$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with $tg^-$ and $tg^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

**Fig. 5** Calculated Raman spectra displayed in the 900-800 $cm^{-1}$ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having a pP-II backbone. (A) conformers with $g^-$ and $g^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with $g^+$ and $g^-$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with $tg^-$ and $tg^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).
with solid and dashed lines, respectively. (C) conformers with $tg^-$ and $tg^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

**Fig. 6** Calculated Raman spectra displayed in the 900-800 cm⁻¹ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having a classic γ-turn backbone. (A) conformers with $g^-g^-$ and $g^-g^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with $g^+g^-$ and $g^+g^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with $tg^-$ and $tg^+$ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

**Fig. 7** 2D graphic representations showing the affine function ($v_{scal}=a v_{calc}+b$) used for scaling the calculated wavenumbers. $v_{scal}$: scaled wavenumber (cm⁻¹), $v_{calc}$: raw calculated wavenumber (cm⁻¹) obtained from the lowest energy conformer. In each graph, empty circles have the coordinates ($v_{obs}$, $v_{calc}$) along the vertical and horizontal axes, where $v_{obs}$ is the observed wavenumber (cm⁻¹).

(A) Based on the DFT calculations at the B3LYP/6-311++G(d,p) level. (B) Based on the DFT calculations at the M062X/6-311++G(d,p) level. (C) Based on the DFT calculations at the ωB97XD/6-311++G(d,p) level. The constants (a,b) as determined by a linear regression are reported on each graph. Correlation constant r>0.995 in all cases.

**Fig. 8** Comparison between the observed and calculated Raman spectra of the cationic species of the tripeptide Gly-Tyr-Gly in the middle wavenumber spectral region. (A) Observed Raman spectrum in H₂O samples as taken from Fig. 2C. (B) Calculated Raman spectrum issued from the DFT calculations by means of B3LYP functional. (C) Calculated Raman spectrum issued from the DFT calculations by means of M062X functional. (D) Calculated Raman spectrum issued from the DFT calculations by means of ωB97XD functional. See Section 3.6 for details concerning the calculation of Raman spectra.

**Fig. 9** Calculated Raman spectra representing conformers with four different backbone secondary structures (extended chain, helix, pP-II and classic γ-turn) and corresponding to one of the three possible $g^-, g^+$ and $t$ orientations of the side chain $\chi_1$ torsion angle. All spectra are issued from the DFT calculations by means of B3LYP functional. See section 3.7 for details concerning the calculation of Raman spectra. The frame drawn with broken line delimit the spectral region in which the Tyr Raman doublet is observed. $\rho$ represents the intensity ratio of the Tyr Raman doublet.
<figure><img src="image_2.png" /><figcaption>**Fig. 1** Structural parameters of the cationic species of the tripeptide Gly-Tyr-Gly. (A) Backbone torsion angles defined along the chemical bonds in going from N<sup>ter</sup> (left) to C<sup>ter</sup> (right) terminal groups. (B) Torsion angles defined along the side chain chemical bonds. χ<sub>2</sub>(g<sup>+</sup>) and χ<sub>2</sub>(g<sup>-</sup>) orientations are presented in (A) and (B), respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>**Fig. 2** Raman spectra recorded in the 1800-600 cm⁻¹ spectral region. (A) Raw Raman spectrum obtained from the samples of Gly-Tyr-Gly dissolved in H₂O (20 mM, pH 6.5). Red asterisk indicates the position of the most intense band assigned to TFA. (B) Buffer (H₂O) spectrum. (C) Buffer (H₂O) and TFA subtracted spectrum of the tripeptide Gly-Tyr-Gly; (D) Buffer (D₂O) and TFA subtracted spectrum of the tripeptide Gly-Tyr-Gly. The frame drawn with broken line delimit the spectral region in which the Tyr characteristic doublet and its satellite bands are observed.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**Fig. 3** Calculated Raman spectra displayed in the 900-800 cm⁻¹ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having an extended chain backbone. (A) conformers with g⁻g⁻ and g⁻g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with g⁺g⁻ and g⁺g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with tg⁻ and tg⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).</figcaption></figure>
**Fig. 4**
Calculated Raman spectra displayed in the 900-800 cm⁻¹ spectral region and Grapdic representation of the conformers of Gly-Tyr-Gly having

a helical backbone. (A) conformers with g⁻g⁻ and g⁻g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with g⁺g⁻ and g⁺g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with tg⁻ and tg⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).

<figure><img src="image_5.png" /></figure>
Fig. 5

Calculate the Raman spectrum of the α-glycyrhamidine G (Gly-Tyr-Gly) in the solid state, and study its spectral region and Gr magnetic resonance structure of the conformers of

Gly-Tyr-Gly having a pP-II backbone. (A) conformers with *g*<sup>-</sup>*g*<sup>-</sup> and *g*<sup>+</sup>*g*<sup>+</sup> side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with *g*<sup>+</sup>*g*<sup>-</sup> and *g*<sup>+</sup>*g*<sup>+</sup> side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with *tg*<sup>-</sup> and *tg*<sup>+</sup> side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), and hydrogen (white).

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>**Fig. 6** Calculated Raman spectra displayed in the 900-800 cm⁻¹ spectral region and Graphic representation of the conformers of Gly-Tyr-Gly having a classic γ-turn backbone. (A) conformers with g⁻g⁻ and g⁻g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (B) conformers with g⁺g⁻ and g⁺g⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. (C) conformers with tg⁻ and tg⁺ side chain orientations and their Raman spectra drawn with solid and dashed lines, respectively. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white).</figcaption></figure>
<figure><img src="image_8.png" /></figure>

**Fig. 7** 2D graphic representations showing the affine function ($v_{scal}=a v_{calc}+b$) used for scaling the calculated wavenumbers. $v_{scal}$: scaled wavenumber (cm$^{-1}$), $v_{calc}$: raw calculated wavenumber (cm$^{-1}$) obtained from the lowest energy conformer. In each graph, empty circles have the coordinates ($v_{obs}$, $v_{calc}$) along the vertical and horizontal axes, where $v_{obs}$ is the observed wavenumber (cm$^{-1}$).

(A) Based on the DFT calculations at the B3LYP/6-311++G(d,p) level. (B) Based on the DFT calculations at the M062X/6-311++G(d,p) level. C) Based on the DFT calculations at the ωB97XD/6-311++G(d,p) level. The constants (a,b) as determined by a linear regression are reported on each graph. Correlation constant r>0.995 in all cases.
<figure><img src="image_9.png" /><figcaption>**Fig. 8**
Com Paris on betwe en the obser ved and calcul ated Rama n spectra of the catio nic speci es of the tripep tide Gly- Tyr- Gly in the middl e wave numb er spectr al</figcaption></figure>

region. (A) Observed Raman spectrum in H₂O samples as taken from Fig. 2C. (B) Calculated Raman spectrum issued from the DFT calculations by means of B3LYP functional. (C) Calculated Raman spectrum issued from the DFT calculations by means of M062X functional. (D) Calculated Raman spectrum issued from the DFT calculations by means of ωB97XD functional. See Section 3.6 for details concerning the calculation of Raman spectra.
<figure><img src="image_10.png" /><figcaption>**Fig. 9** Calculated Raman spectra representing conformers with four different backbone secondary structures (extended chain, helix, pP-II and classic γ-turn) and corresponding to one of the three possible g-, g+ and t orientations of the side chain χ₁ torsion angle. All spectra are issued from the DFT calculations by means of B3LYP functional. See section 3.7 for details concerning the calculation of Raman spectra. The frame drawn with broken line delimit the spectral region in which the Tyr Raman doublet is observed. ρ represents the intensity ratio of the Tyr Raman doublet.</figcaption></figure>