# Conformation Dependence of Phenylalanine Characteristic Raman Markers

Belén Hernández, Yves-Marie Coic, Sergei G. Kruglik, Santiago

Sanchez-Cortes, Mahmoud Ghomi

► To cite this version:

Belén Hernández, Yves-Marie Coic, Sergei G. Kruglik, Santiago Sanchez-Cortes, Mahmoud Ghomi. Conformation Dependence of Phenylalanine Characteristic Raman Markers. Journal of Raman Spectroscopy, 2024, 56 (3), pp.262-277. 10.1002/jrs.6759. hal-05090550

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Distributed under a Creative Commons Attribution 4.0 International License
Conformation Dependence of Phenylalanine Characteristic Raman Markers

Belén Hernández,ª Yves-Marie Coïc,ᵇ Sergei G. Kruglik,ᶜ Santiago Sanchez-Cortes,ᵈ
Mahmoud Ghomi d*

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

ᵇInstitut Pasteur, Université Paris Cité, CNRS UMR 3523, 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
**Abstract**

Conformational dependence of six characteristic phenylalanine (Phe) Raman markers ($F_i$, i=1,...,6) was assessed through a multiconformational approach. Herein, aqueous solution off-resonance Raman spectra of the cationic species of the tripeptide Gly-Phe-Gly, with amine/amide backbone terminal groups, accompanied by the density functional theory calculations, using three different hybrid functionals (B3LYP, wB97XD and M062X), were presented. A set of 15 initial conformers, having five types of backbone secondary structures (β-strand, polyproline-II, helical, classic and inverse γ-turn) and three plausible side chain orientations, were submitted to geometry optimization. The optimized conformers permitted analyzing the effect of non-bonded interactions, such as intramolecular hydrogen bonds, as well as those occurring through space between aromatic side chain and adjacent peptide bonds. It has been shown that M062X was the only functional capable of maintaining the stability of all conformers, especially those having an inverse γ-turn backbone. In all calculations, extended chains (β-like) and classic γ-turn structures were shown to form the lowest and the highest energy conformers, respectively. Helical and pP-II structures belong to intermediate energy conformers. Raman spectrum was calculated by the thermal (Boltzmann) average of those spectra arising from the whole optimized conformers. An increasing global improvement of the spectral shape is to be noticed in going from the data obtained B3LYP to ωB97XD and M062X. Furthermore, explicit hydration of conformers improves the quality of the calculated spectrum by means of M062X. Wavenumber dispersion of the characteristic Raman markers revealed that $F_5$ (~1004 cm⁻¹) has the lowest dispersion (≤1 cm⁻¹), remaining unaffected by explicit hydration. In contrast, $F_3$ (~1207 cm⁻¹) shows the largest shift (≤6 cm⁻¹), whereas a moderate one (≤3 cm⁻¹) was predicted for $F_1$ (~1605 cm⁻¹), $F_2$ (~1586 cm⁻¹) and $F_4$ (~1035 cm⁻¹). Explicit versus implicit hydration seems to affect the wavenumber dispersion of $F_6$ (~622 cm⁻¹). It can be concluded that the conformational sensitivity of the Phe characteristic markers basically arises from the nature of their vibrational motions.

**Keywords:** Phenylalanine; Tripeptide; Glycylphenylalanylglycine; Extended chain; Polyproline-II; Helix; γ-turn; Solution Raman spectra; Density functional theory calculations
Introduction

Conformational flexibility of peptide chains, particularly in relation with their aggregation
capabilities to form attractive self-assemblies becomes an active field of research in biology,
medicine and material science. [1-23] Numerous investigation tools have been developed,
among which, optical spectroscopy, using either the light absorption, (Electronic circular
dichroism (ECD), Fourier-transform-Infrared absorption (FT-IR), Vibrational circular
dichroism (VCD)), or the light scattering (off- and on-resonance Raman effects, Raman
optical activity (ROA)), [24-42] were shown efficient regarding their wealth of structural
information, as well as their remarkable rapidity in both acquisition and post-treatment. Based
on the modulation of the electronic polarizability at the pace of nuclear vibrational motions,
inelastic light (Raman) scattering is particularly appreciated because of the strong
characteristic lines arising from the aromatic residues. [43-45] Beyond their use in traditional
vibrational spectroscopy on peptides and proteins, aromatic markers have progressively been
taken as spectral fingerprints in living cell imaging by means of Raman microscopy. [46,47]

Joint experimental and theoretical studies on the zwitterionic species of three aromatic
amino acids (Phe, Tyr, Trp) in aqueous environment, [43,48,49] have brought clarification on
aromatic Raman marker. All located in the middle wavenumber spectral region, these markers
(Table 1) were referred to as Fᵢ (i=1, ..., 6) in Phe, Yᵢ (i=1, ..., 7) in Tyr, and Wᵢ (i=1, ..., 8), in
Trp. Nevertheless, subsequent studies on Tyr and Trp characteristic markers [50,51] have
revealed the insufficiency of free amino acids for analyzing the conformational dependence of
Raman markers observed in a peptide chain. This is mainly due to the expected π-π
interactions, occurring through space between an aromatic ring and its two adjacent peptide
(-CO-NH-) bonds upon aromatic side chain rotation (Fig. 1B,D). Likely, in a dipeptide model
compound, these nonbonded interactions cannot be thoroughly analyzed because of its
truncated N'ter or C'ter group. Consequently, a tripeptide with an aromatic residue placed at its
middle (second) position (Fig. 1A), becomes naturally the shortest motif to be considered. A
phenomenological methodology, using aqueous solution optical spectroscopic data was
developed to probe the structural features of tripeptides. [52,53] It led to the conclusion that
despite a widely spread idea, a tripeptide backbone is not completely disordered, adopting
possibly several ordered secondary structures, such as β-strand, polyproline-II (pP-II), helical
and γ-turn folds. In this framework, optical and NMR data collected from the Gly-rich
tripeptides, Gly-Xxx-Gly (where XXX= natural amino acid other than Gly), [54, 55] were shown
the coexistence of β-like and pP-II as major populations in solution, with some subtle
differences between the relative proportions of these structures, depending on the nature of
the middle residue (Xxx) chemical nature. [56] The influence of the two terminating residues
on the conformational landscape was also stressed upon comparison of the structural features
of Gly-Phe-Gly and Ala-Phe-Ala. [55] Both tripeptides self-aggregate upon increasing
concentration in solution, presumably favored by stabilizing π-π interactions between Phe
residues. [55]

The last three decades have also seen the emergence of sophisticated computational
methods, revealing the sequence-dependent conformational features of peptide chains.
Structural modeling based on homology unveiled the inter-dependence of the backbone
structure (determined by successive (Φ,ψ) pairs of torsion angles along the peptide backbone)
and the side chain orientation (defined by successive χᵢ torsion angles) of the constituting
amino acid residues. [57] Statistical analysis of the conformational data of residues using the
protein X-ray and NMR data bank, [58-60] permitted structural prediction of a number of
unfolded polypeptides. Force-field-based molecular dynamics was used to sample the
conformational preference of the residues involved in a peptide chain. Within nanosecond
time-scale, the propensity of the two Phe containing tripeptides, Gly-Phe-Gly and
Ala-Phe-Ala, surrounded by a large number (n) of hydrating water molecules, representing
several hydration layers, [55] to form β-like, pP-II, helical and γ-turn structures, was analysed.
With the progress in quantum mechanical calculations, especially those based on the density
functional theory (DFT) approach, [61] attention was focused on the structural flexibility of
implicitly and explicitly hydrated peptides. In this framework, one can cite the previous
calculations devoted to β-like and pP-II structural stabilities on (i) a simplified model
compound (referred to as alanine dipeptide) surrounded either by n=4 explicit water
molecules, [62] or by a variable hydration number (n=0-to-12); [63] (ii) the protonated
Ala-Ala-Ala hydrated by n=0-to-37 water molecules; [64] and (iii) the protonated Gly-Xxx-
Gly (where XXXX=Ala, Leu, Ile), interacting with n=10 water molecules. [65] From these
calculations, it appeared that upon increasing hydration number a higher stability for pP-II
conformer can be expected in comparison with a β-like one. More recent DFT calculations
attempted to perform a systematic investigation on both structural and vibrational
(wavenumbers and Raman intensities) features of tripeptides. [50,51,66,67] Attempt was made to
find suitable functionals, reproducing as accurately as possible the observed data from the
selected tripeptides. Raman intensities were calculated in the framework of a
multiconformational analysis, assuming that the observed Raman intensity of a tripeptide
corresponds to the thermal (Boltzmann) average of those arising from its conformers. As a
consequence, a good agreement with the observed spectrum of a peptide can be achieved
when the selected theoretical level provides accurate relative energies and vibrational data for
the conformers of a peptide. In the case of the structurally simplest tripeptide, Gly-Gly-Gly, it
was shown that the relative energy values obtained by DFT calculations are generally
exaggerated in comparison with those provided by the second-order perturbative Møller-
Plesset (MP2) method, taken as reference. [68] Nevertheless, the best theoretical data often
correspond to double-exchange, dispersion corrected functionals, such as M062X [69] and
ωB97XD, [70-72] as compared to a traditional hybrid functional such as B3LYP. [73,74] As far as the DFT application to the aromatic containing tripeptides are concerned, the results relative to Gly-Tyr-Gly [50] and Gly-Trp-Gly, [51] showed the remarkable influence of both aromatic side chain orientation, and backbone structure on the well-known characteristic Raman doublets observed at ~850-830 cm⁻¹ in Tyr (Y₅-Y₆ doublet) and at ~1360-1340 cm⁻¹ in Trp, (W₄-W₅ doublet), both arising from the fundamental modes of the phenol and indole rings, respectively. Nevertheless, no consistent information on the conformational sensitivity of the Phe characteristic Raman markers has been made available up to now. To bring clarification on this point, we present here a multiconformational analysis on the tripeptide Gly-Phe-Gly, combining aqueous solution off-resonance Raman spectra with DFT structural and vibrational data.

# Methods

## Chemical synthesis

The tripeptide, NH₂-Gly-Phe-Gly-CONH₂, with amine/amide backbone terminal groups, was synthesized at the Institut Pasteur (Paris, France) according to the Fmoc/tBu solid-phase strategy [75] from a Rink amide resin on an ABI 433 synthesizer (Applied Biosystems, Foster City, CA). Details concerning the synthesis procedure was recently reported. [50] The final net peptide content was determined by quantitative amino acid analysis on a Hitachi L8800 apparatus, giving overall isolated yield of 72%. The purity control of the final peptides (>98%) was performed by RP-HPLC on an Aeris Peptide 3,6µm XB-C18 analytical (2.1x100 mm) column (Phenomenex, Le Pecq, France), using a linear gradient of acetonitrile in 0.08% aqueous TFA over 20 min at a 0.35 mL/min flow rate. Electrospray ionization mass spectrometry was carried out on a quadrupole-TOF Micro mass spectrometer (Waters) equipped with a Z-spray API source. The experimental masses were acquired in positive-ion
mode and were consistent with the theoretical isotopic values (expected M+H⁺ 279.1452, observed 279.1696).

## Solution samples

Solution sample containing the tripeptide with trifluoroacetic acid (TFA) as counterion, was prepared at room temperature by dissolving lyophilized powder in water taken from a Millipore filtration system. The final concentration was 20 mM (5.6 g/L) leading to a good signal/noise ratio. Upon dissolution pH was 6.5±0.1. As a result, because of the high amine pKₐ value (~10), the cationic species with NH₃⁺/CONH₂ terminal groups (Fig. 1A) was the major species in solution. H-D isotopic substitution on labile hydrogen atoms involved in the backbone NH chemical bonds, was achieved by dissolving tripeptide in D₂O (100% purity) purchased from Eurisotop (Saclay, France).

## Raman spectroscopy

50 µL solution samples 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. Room temperature 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. Buffer subtraction and smoothing of the observed Raman spectra could be performed by using 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).

Initial conformers for DFT calculations

Previous molecular dynamics (MD) simulations on Gly-Phe-Gly and Ala-Phe-Ala tripeptides
[55] have shown the possible presence of five types of the ordered secondary structures, such
as β-like, pP-II, helical, classic and inverse γ-turn, having different populations. Same
backbone secondary structures were considered in the present work. Seven torsion angles (ψ₁,
ω₁, Φ₂, ψ₂, ω₂, Φ₃, ψ₃) permit determination of the backbone secondary structure, where
subscripts recall the residue numbers (Gly¹, Phe² and Gly³) (Fig. 1A). ω, defined around the
peptide bond (-CO-NH-), remains close to 180°. Their initial rotamers were constructed using
the pairs (Φ,ψ)=(-135°,+135°) for β-strand, (Φ,ψ)=(-75°,+150°) for pP-II, and
(Φ,ψ)=(-60°,-45°) for α-helix. Classic and inverse γ-turn folds, were supposed to be centered
on the middle residue (Phe²), and 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. [76,77] All
other backbone torsion angles in a given γ-turn conformer, i.e., those relative to Gly¹ and Gly³
were initially set to those relative to a β-strand.

Two other conformational angles, (χ₁, χ₂) (Fig. 1A), precise the aromatic side chain
orientation (Phe²). χ₁(N-Cα-Cβ-Cγ), defined around the single bond (Cα-Cβ), can naturally
adopt three privileged orientations, namely gauche⁺ (or g⁺) (60°±60°), gauche⁻ (or g⁻)
(-60°±60°), and trans (or t) (180°±60°). In contrast, because of the sp² type of Cγ atom located
in the phenyl ring, χ₂ defined around Cβ-Cγ bond, is limited to two privileged orientations,
i.e., g⁺(90°±90°) and g⁻(-90°±90°). Moreover, because of the indiscernibility of Cδ₁ and Cδ₂
atoms (phenyl ring), two possible definitions can be envisaged for $\chi_2$, either $\chi_2(C_\alpha-C_\beta-C_\gamma-C_{\delta1})$ or $\chi_2(C_\alpha-C_\beta-C_\gamma-C_{\delta2})$. One of these two angles is always positive ($g^+$), and the other one negative ($g^-$), while the absolute value of their difference remains close to 180°. For the sake of clarity, both possible values assignable to $\chi_2$ were reported in Tables S1-S4 (Supporting Information), and the $\chi_2$ orientation was correspondingly referred to by the special symbol $g^\pm$. As a consequence, three distinct side chain orientations, i.e., $g^+g^\pm$, $g^-g^\pm$ and $tg^\pm$, with respect to the ($\chi_1, \chi_2$) angles, were considered. The initial values given to $\chi_1$ were +60° ($g^+$) and -6° ($g^-$) and 180° (t), whereas those assigned to $\chi_2$ were +90° ($g^+$) and -90° ($g^-$). It should be emphasized that the presently considered initial Phe side chain rotamers reflect somehow those relative to the maxima of the probability densities of $\chi_1$ and $\chi_2$ torsion angles, as derived from the statistical analysis of the amino acid residues involved in polypeptide chains. [60]

Considering the five possible backbone rotamers, and the three possible side chain orientations, a total number of 5x3=15 initial conformers were thus geometry optimized by means of each functional.

## Hydration models used in DFT calculations

The effect of both implicit and explicit hydration on the structural and vibrational data of the tripeptide conformers, was analyzed. In a first step, DFT calculations by means of each selected functional were carried out on all the 15 conformers embedded in a polarizable continuum medium (PCM), [78,79] having a relative permittivity corresponding to bulk water ($\epsilon_r=78.39$). In a second step, with the best functional (ensuring the structural stability of all 15 conformers) a mixture of implicit and explicit hydration models was considered. In this approach, a given conformer with its surrounding water molecules were placed in the aforementioned solvent continuum. To choose a reasonable hydration number ($n$), we were inspired from the former DFT calculations on amino acids [48,49] and tripeptides, [66] performed
with a minimal hydration number, ensuring H-bonds with all polar groups. These water
molecules naturally belong to the first hydration shell. More precisely, the considered solvent
continuum is assumed to be capable of reproducing accurately the effect of the second and
higher hydration layers. This protocol avoids the formation of H-bonds between water
molecules, and as a result, the compensation of the molecular internal energy due
conformational changes by the energy of interaction between surrounding water molecules, is
avoided. A water molecule can play simultaneously the role of the H-bond donor and
acceptor. Consequently, four water molecules were found to be sufficient to hydrate the
adjacent N-H and C=O bonds along the tripeptide chain. As to the phenyl ring hydration, a
pioneering work on benzene [80] had revealed that water molecule can bind to benzene through
the so-called Hw...π interaction (where Hw is a water hydrogen atom interacting with the
π-electron cloud of an aromatic ring). Following this idea, a theoretical work was performed
on four amino acids (Phe, Tyr, Trp, His), in order to find possible locations of water
molecules that can bind to their aromatic rings. [81] Concerning Phe, it was shown that only
one water molecule can be placed onto its phenyl ring. Therefore, a total hydration number of
n=5 was chosen to hydrate, in a minimalist way, both the backbone and the side chain of
Gly-Phe-Gly conformers.

**Choice of suitable hybrid functionals in DFT calculations**

A recent work [67] permitted the comparison of the energetic and structural data obtained from
using eleven different functionals. It appeared that ωB97XD was the only functional capable
of preserving the structural stability of all considered conformers. ωB97XD has also provided
suitable Raman intensities in the case of Gly-Trp-Gly. [51] Moreover the performances of
M062X functional in the DFT calculations on the cationic species of four tripeptides
Gly-Gly-Gly, Gly-Ala-Gly, Ala-Ala-Ala and Ala-Phe-Ala, were also shown. [66] However, the
routinely used functional B3LYP gave accurate theoretical data in the case of Gly-Tyr-Gly.

[50] As a consequence, in order to be able to choose the adequate functional in the case of Gly-Phe-Gly, we compared the energetic, structural and vibrational data provided by B3LYP and ωB97XD and M062X.

## DFT calculations and post-treatment of theoretical data

Each initial conformer of Gly-Phe-Gly was geometry optimized using one of the three used functionals, and the polarized triple-zeta Gaussian atomic basis set, equipped with diffuse functions, referred to as 6-311++G(d,p). [82] Theoretical calculations were performed by means of the Gaussian16 package. [83] Each geometry optimization was followed by harmonic vibrational calculations. The correspondence of the optimized geometry to a local minimum was proved by the absence of any imaginary frequency.

In a previous experimental investigation based on the solution ECD and ¹H-NMR data derived from the tripeptides, having the generic sequence Gly-Xxx-Gly (where XXX is a natural amino acid), the enthalpic and entropic contributions to the free energy, were determined through a two-state (β-like/pP-II) conformational transition. [84] It has been evidenced that the results depend on the middle residue (Xxx) chemical nature. In the present DFT calculations, the assessment of relative energies was based on the total energy (E<sub>tot</sub>) of the optimized conformers, where E<sub>tot</sub>=E<sub>c</sub>+ΔG, i.e., the sum of electronic energy (E<sub>e</sub>) and free energy correction (ΔG). As a result, each optimized conformer was characterized by its relative energy (ΔE), as compared to that of the lowest energy conformer, for which ΔE was set to zero.

Assignment of the vibrational modes was made with the calculated force constants and atomic displacements, leading to the evaluation of the so-called potential energy distribution (PED), [85] as expressed in terms of internal coordinates, i.e., bond stretching (v), angular
bending (δ), out-of-plane bending (ω), and torsion (τ) coordinates. Raman activities issued
from DFT calculations were converted to Raman intensities following the previously reported
expression. [86] Calculated Raman intensities were obtained through a multiconformational
approach, in which the thermal (Boltzmann) average of the spectra arising from different
conformers, is compared to the observed one in solution samples. To scale the raw calculated
wavenumbers (vcalc), an affine relation of the form: vscal=avcalc+b, was used, where vscal
represents scaled wavenumbers, and (a,b) are constants. The advantages of this scaling
procedure were extensively detailed in our preceding reports on tripeptides. [50,51] However,
the values given to (a,b) constants depend on the used functional. A linear regression
procedure, giving a correlation coefficient>0,995, provided the values (a,b)=(0.9836,4.115),
(a,b)=(0.9354,46.08), and (a,b)=(0.9744,11.19) relative to the wavenumbers issued from the
calculations by means of B3LYP, ωB97XD and M062X functionals, respectively.

**Results and discussion**

Raman spectra from native and deuterated species

Raw Raman spectrum recorded from the H₂O solution samples of the tripeptide, as well as
that from the buffer, are displayed in Fig. 2A within the 1800-600 cm⁻¹ region. The details
concerning the spectral features of the counterion (TFA), and its subtraction from the
observed spectra, were reported previously, [27,50] giving rise to the treated spectrum shown in
Fig. 2B. Likely, the Raman spectrum arising from D₂O samples is displayed in Fig. 2C. The
most important spectral features of the observed Raman spectra can be remarked as follows:
(i) amide-I vibrations is characterized by a weak and broad band peaking at 1691 cm⁻¹ (Fig.
2B), downshifted to 1676 cm⁻¹ in D₂O (Fig. 2C); (ii) the six Phe characteristic Raman
markers, referred to as Fᵢ (i=1,...,6), marked in red color in Fig. 2B, present wavenumber
downshifts not exceeding 1 cm⁻¹. Noteworthy, the classical Raman spectrum of the
zwitterionic species (with NH₃⁺/COO⁻ terminal groups) of the same tripeptide obtained from solution samples (40 mM, pH 7), was previously published. [83] Only slight wavenumber shifts of the characteristic Phe Raman markers, not exceeding 5 cm⁻¹, can be noticed in going from cationic to zwitterionic species of the tripeptide.

Structural and vibrational data obtained upon implicit hydration

Both B3LYP and ωB97XD functionals were unable to stabilize inverse γ-turn conformers. This situation has also been encountered in the recent DFT calculations on Gly-Tyr-Gly [50] and Gly-Trp-Gly. [51] As a result, only M062X was capable of providing a complete set of 15 optimized conformers relative to the five considered secondary structures.

Tables S1-S3 (Supporting Information) give the relative energies, as well as the conformational (torsion) angles of the optimized conformers, together with the lengths of intramolecular H-bonds, as derived from the calculations performed by means of B3LYP, ωB97XD and M062X functionals, respectively. To discuss the theoretical data, we preferred to keep together in all Tables and Figures the conformers belonging to the same secondary structure, and present them in the order g⁺g⁺, g⁻g⁺ and tg⁺ of their Phe² orientations. This facilitates the comparison of the obtained data from one conformer to another, and from one functional to another. Below, we describe the most striking effects encountered in the calculated data.

Whatever the used functional, all the calculated conformers fall within a ~8 kcal/mol relative energy interval, and the lowest energy conformer (ΔE=0) always corresponds to a β-like backbone structure. These conformers, also referred to as “extended chains”, are characterized by their absolute value of backbone torsion angles close to 180°, giving rise to a quasi-planar stretched backbone (Fig. 3A). However, the influence of the side chain
orientation on the backbone structure, is worth of mentioning, especially on (Φ₂,ψ₂) corresponding to Phe². As a result, the absolute values of (Φ₂,ψ₂) relative to g⁺g⁺ and tg⁺ orientations, are found close to those generally assigned to a β-strand (~135°). To bring an explanation to this effect, we should remind that the location of the aromatic ring with respect to the backbone depends primarily on χ₁ torsion angle. More precisely, with a χ₁(g⁻) orientation, the aromatic ring is oriented toward the backbone N'ter side (Fig. 1B), while with a χ₁(t) it will be found on its C'ter side (Fig. 1D). Obviously, a χ₁(g⁺) places the aromatic ring between the aforementioned situations (Fig. 1C). The role of the second side chain torsion angle χ₂(g⁺) is to maintain stacked the aromatic ring with the backbone chemical groups. Therefore, the interactions occurring through space, between the phenyl ring and its adjacent peptide bonds, might affect not only the relative energies, but also the geometrical parameters, and preferentially the backbone torsion angles of Phe². The role of the functional, and its inherent capacity to treat nonbonded interactions, is also to be emphasized. For instance, regarding the lowest energy conformer (ΔE=0), while B3LYP and M062X assigns a tg⁺ side chain orientation to its middle residue, ωB97XD provides to Phe² a g⁺g⁺ orientation (Tables S1-S3, Supporting Information).

In contract, pP-II, helical and classic conformers are well preserved upon geometry optimization (Figs. 3B, 4A,C), especially in the case of the calculated data with ωB97XD and M062X (Tables S2 and S3, Supporting Information), see the backbone torsion angles (Φ₂,ψ₂) assigned to Phe². In all cases, the influence of the side chain orientation on the torsion angles, as well on the relative energy of conformers are to be noticed, without neglecting the effect of the functional on their relative energies. Classic γ-turn conformers are associated to the highest ΔE values, falling within the 3-8 kcal/mol interval. As mentioned above, inverse γ-turn conformers could only be stabilized with M062X (Table S3, Supporting Information).
Interestingly, this functional places inverse γ-turn conformers among those with intermediate relative energies.

Folded structures, such as helical and γ-turn structures, manifest an intramolecular H-bond (Fig. 4). In a helical structure (Fig. 5A), the H-bond is formed between the backbone C=O (Gly¹) and one of the hydrogen atoms involved in the C¹er amide group (Fig. 4A). This short contact plays somehow that of the so-called i→i+4 H-bond in a longer α-helix. [⁵⁷] In a γ-turn, the intramolecular H-bond, also referred to as “turn closing H-bond”, is formed between the backbone C=O (Gly¹) and N-H (Gly³) (Fig. 4B,C). Beyond the fact that the length (d<sub>HB</sub>) of intramolecular H-bonds is shown to be functional dependent (Tables S1-S3, Supporting Information), they are also affected by both the backbone structure and the side chain orientation. For instance, while all functionals assign the shortest d<sub>HB</sub> value to the classic γ-turn conformer, having a Phe² residue in g⁺g⁻ orientation, its length is estimated to be 1.92 Å by B3LYP, 1.80 Å by ωB97XD, and 1.90 Å by M062X. Surprisingly, this conformer corresponds to the highest calculated relative energy. This leads us to conclude that the length of an intramolecular H-bonds is not always an indicator of its relative energy in a tripeptide, where the favourable (versus unfavourable) nonbonded interactions between the aromatic ring and backbone play a key role in forming the energy landscape.

To give a schematic picture of the conformational landscape brought about by the present DFT calculations in a solvent continuum, Fig. 5 displays the histogram representing the normalized populations (in percent) of each backbone type (extended chain, pP-II, helix, classic and inverse γ-turn) based on their thermal (Boltzmann) weights. Irrespective of the used functional, extended chains form the largest population (ranging between 73% with B3LYP, and 81% with ωB97XD) of the total population, owing to their low relative energies (Fig. 5A-C). The influence of the functional appears on the second rank population. While B3LYP places pP-II conformers at the second rank (25%, Fig. 5A), ωB97XD and M062X
provide this rank to helical conformers (17%, Fig. 5B,C). Interestingly, because of their high
ΔE values, a negligible population is conferred to classic γ-turn structures by means of all
functionals (Fig. 5A-C).

Construction of the thermal averaged Raman spectrum of the tripeptide by means of the
vibrational data (wavenumbers and Raman intensities) obtained with M062X, is reported in
Fig. 6. The calculated Raman spectra relative to each of the 15 calculated conformers, with
pondered intensities obtained using their Boltzmann weights, are displayed. Summation of
these spectra gives rise to the thermal averaged spectrum, referred to as “sum”, displayed on
the top of Fig. 6. As it can be seen, only the spectra corresponding to the lowest energy
conformers, i.e., having a ΔE<2 kcal/mol, can bring a significant contribution to the sum
spectrum. The thermal averaged Raman spectra using the three functionals are reported in Fig.
7C-D, and compared to the observed one (Fig. 7A). It is a matter of fact that the calculated
Raman spectra provided by different functionals account for the six characteristic Phe
markers (Fᵢ, i=1, ..., 6). Nevertheless, all of them underestimate the relative intensity of the
strongest Raman marker (F₅ at ~1004 cm⁻¹). While B3LYP provides the closest wavenumbers
to the observed ones, an increasing global improvement of the spectral shape, can be noticed
in going from B3LYP (Fig. 7B) to ωB97XD (Fig. 7C) and M062X (Fig. 7D).

Table S5 (Supporting Information) shows the comparison between the observed and
calculated wavenumbers accompanied by their assignments derived from the vibrational
calculations on the lowest energy conformer (ΔE=0) obtained by means of M062X (Fig. 3A).

For further use of readers, Tables S6-S10 (Supporting Information) provide the atomic
cartesian coordinates of the 15 optimized conformers optimized in continuum by means of
M062X functional.

Structural and vibrational data obtained upon explicit hydration
In the preceding Section, we mentioned the undeniable advantages of M062X in providing a
complete set of 15 optimized conformers in a solvent continuum, as well as in improving the
general shape of the calculated Raman spectrum in the middle wavenumber region. Therefore,
it appeared natural to analyze the effect of the explicit hydration with the same functional.
The graphical representation of the optimized conformers, each one surrounded by 5 water
molecules, is displayed in Figs. 8 (extended chains and pP-II) and 9 (helical, inverse and
classic γ-turn). Table S4 (Supporting Information) shows the relative energies and
conformational angles of the explicitly hydrated conformers. Below, we summarize the most
prominent features of the geometry optimized conformers.

The phenyl ring is hydrated by at least one water molecule pointing its hydrogen atoms
toward the aromatic ring, and four water molecules interact with all backbone polar groups. In
addition, no water dimer appeared upon optimization. Relative energies of the conformers
remain below 8 kcal/mol, and the lowest energy conformer (ΔE=0) is an extended chain
conformer (Fig.8A), having a Phe² side chain with g·g'. It is to be reminded that in continuum
a tg' was assigned to the lowest energy conformer (Fig.3A). As described in the last section in
a g·g' orientation, the phenyl ring turns toward N'ter side, thus lowering the molecular energy
by a favourable interaction between the aromatic ring and the backbone positively charged
terminal group (NH₃').

All helical and γ-turn structures remain stable in the presence of explicit water molecules.
This fact can be verified through the values of (Φ₂,ψ₂) torsion angles (Table S4, Supporting
Information). The values of the relative energies are affected by explicit hydration. The
intermolecular H-bonds of the folded conformers remain intact, and their lengths show little
variations (up to 10%) in going from implicit (Fig. 4A) to explicit hydration (Fig. 9A). The
shortest H-bond length (1.91 Å) corresponds to a classic γ-turn having a g⁺g⁺ aromatic side
chain, while the longest one (2.27 Å) is relative to an inverse γ-turn with a tg' orientation. As
in the case of implicit hydration, the highest relative energies are assigned to classic $\gamma$-turn conformers (ranging between 3 and 8 kcal/mol), while those relative to inverse $\gamma$-turn show a net decrease upon using explicit hydration.

Fig. 5D presents the histogram of the populations of different secondary structures. Its comparison with those corresponding to implicit hydration (Fig. 5C) reveals a 10% decrease (from 79% to 71%) concerning the population of extended chains, accompanied by an increase (from 2% to 9%) for inverse $\gamma$-turn and pP-II (from 2% to 4%) conformers. In contrast, the population corresponding to classic $\gamma$-turn structures remains negligible, as in the case of implicit hydration (Fig. 5D).

Thermal averaging of the calculated Raman spectra arising from different explicitly hydrated conformers is presented in Fig. 10. As it can be seen, extended chain, helical and inverse $\gamma$-turn conformers contribute to the construction of the thermal averaged (sum) spectrum displayed on the top. In Fig. 5E the latter spectrum is compared to the observed one (Fig. 5A) and those obtained by implicit hydration (Fig. 5B-D). As it can be seen, the general spectral shape within the whole middle wavenumber region is improved upon considering explicit hydration.

For further use of readers, the atomic cartesian coordinates of the 15 optimized conformers in the presence of 5 water molecules, all embedded in a polarizable continuum, were displayed in Tables S11-S15 (Supporting Information).

## Wavenumber dispersion of characteristic Phe Raman markers

The conformational dependence of the six Phe characteristic Raman markers ($F_i$, i=1,...,6), can be achieved through a statistical analysis of the wavenumbers issued from the vibrational calculations of different conformers (Table 2). Although the average wavenumbers are functional dependent, their standard deviations can guide us to analyze their conformational
dispersion. Interestingly, F<sub>5</sub>, the strongest Raman marker is associated to the lowest dispersion value (≤1 cm<sup>-1</sup>), leading us to conclude that implicit versus explicit hydration does not affect this marker. In contrast, F<sub>3</sub> marker shows the largest shift (3 to 6 cm<sup>-1</sup> depending on the used functional), while a moderate value (≤3 cm<sup>-1</sup>) is found for F<sub>1</sub>, F<sub>2</sub> and F<sub>4</sub>. The special case of F<sub>6</sub>, however, is to be emphasized, presenting a low dispersion (≤2 cm<sup>-1</sup>) in continuum versus a larger one (4 cm<sup>-1</sup>) upon explicit hydration.

Concluding remarks

In continuation of our recent works on the conformational dependence of the Tyr and Trp
characteristic Raman markers, [50,51] our aim in the present report was to assess the sensitivity
of the six Phe characteristic markers (F<sub>i</sub>, i=1, ..., 6). To achieve this objective, a structurally
simple tripeptide, Gly-Phe-Gly in its cationic form, was considered. The peptide backbone
propensity to adopt the ordered structures such as β-strand, pP-II, and γ-turn, as well as the
capability of the aromatic side chain to adopt the three plausible orientations, namely g<sup>+</sup>g<sup>±</sup>, g<sup>-</sup>g<sup>±</sup> and tg<sup>±</sup>, were analysed by DFT calculations. The theoretical vibrational data (wavenumbers
and Raman intensities) gathered from all conformers, were used to calculate the thermal
averaged Raman spectrum, compared to the observed one. It was shown that M062X gives
the best structural and vibrational data, as also evidenced by previous DFT calculations on
other cationic tripeptides. [64,66] In the present work, attempt was made to use a minimal
hydration number, belonging to the first hydration shell, in order to basically emphasize the
effect of the H-bonds with the tripeptide chemical groups, and not those between the
surrounding water molecules. Explicit hydration with five water molecules hydrating the
aromatic ring (through the so-called H<sub>w</sub>...π interaction), as well as the backbone polar groups
(through the H-bonds with N-H, C=O and NH<sub>3</sub><sup>+</sup> groups), improves the quality of the
vibrational data. In this case, the influence of higher hydration layers around the tripeptide
was considered by means of the solvent continuum.

Former classical MD simulations performed on the zwitterionic Gly-Phe-Gly surrounded
by explicit water molecules, were consistent with a possible coexistence of β-like, pP-II,
helical, classic and inverse γ-turn. [55] Examination of the MD trajectories had led to suggest
that the major populations in solution correspond to β-like (40-to-69%) and pP-II (29-to-42%)
conformers, while other backbone structures constitute minor populations in solution. In
contrast, the present DFT calculations refer to β-like conformers a higher population (between
71 and 81%), and a comparatively lower one to pP-II conformers (between 4 and 25%). A
non-negligible population (2-to-17%), however, was assigned to helical conformers,
accompanied by the prediction of very low, even negligible, γ-turn structures. In fact, the
tripeptide used in the present work, contains amine/acid (NH₂/CONH₂) backbone terminals.
The major part of the already published observed and calculated data were collected from an
amine/acid (NH₂/COOH) backbone, either in its zwitterionic, or in its cationic (protonated)
form. [55,56,64,66,87] Because of the short length of a tripeptide, the influence of the backbone
terminal groups on the structural and vibrational features of the middle residue, cannot be
omitted. [67] Further NMR data collected from the tripeptide sequence chosen here, would be
needed for comparison to the calculated ones, using preferentially the atomic coordinates
issued from the present optimized conformers (Supplementary Information). It is also worth
emphasizing that the herein used tripeptide sequence contains somehow three amide (-CO-
NH-) bonds, two of them are inter-residue amides, while the third one is located in the C¹er-
terminal group. Therefore, the previous conclusions based on amide I/amide I' band [67]
analysis cannot be entirely applied to it. Obviously, future DFT calculations should also
account for the backbone structural equilibrium upon increasing hydration number.
The conformational sensitivity of the Phe characteristic markers is mainly due to the nature and localization of their vibrational motions. Precisely, F₁, F₂, F₄ and F₅ markers, of which the wavenumbers present either a low or a moderate dispersion (≤3 cm⁻¹), correspond to in-phase vibrational motions entirely localized inside the aromatic ring, whereas F₃, giving the largest shift (≤6 cm⁻¹), involve both the vibrational motions of the phenyl ring (involving its Cδ₁, Cδ₂ and Cγ atoms), as well those close to Cβ atom, increasing naturally its sensitivity to the side chain conformational transitions. [48,49] At last, the behavior difference between F₅ and F₆, is worth emphasizing. Although both markers arise from the phenyl ring breathing modes, their homothetic vibrational motions do not occur with respect to the same molecular axis. [48,49]

**Conflicts of interest**

None of the authors have a conflict of interest to disclose

**Acknowledgements**

The theoretical calculations were granted access to the HPC resources of IDRIS made by Grand Equipment National de Calcul Intensif. The authors thank Dr. Fernando Pflüger for his suggestions on structural analysis.

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**Figure captions**

**Fig. 1** Chemical structure and conformational features of the cationic species of the tripeptide Gly-Phe-Gly.

(A) Definition of the nine torsion angles determining the backbone and side chain conformations. The torsion angle $\chi_1$ defined around the side chain $C_\alpha-C_\beta$ bond in $g^+$ (B), $g^-$ and $t$ (D) orientations. $\pi-\pi$ type interactions between the phenyl ring and the adjacent peptide bonds are reinforced by $\chi_1(g^+)$ and $\chi_1(t)$ orientations. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white). “+” sign designates the electric charge borne by the terminal $NH_3^+$ group.

**Fig. 2** Room temperature Stokes Raman spectra of the cationic species of the tripeptide Gly-Phe-Gly observed in aqueous samples.

(A) Raw Raman spectrum recorded from the aqueous samples of the tripeptide (blue) and buffer ($H_2O$) (green). (B) Treated Raman spectrum of the tripeptide after subtraction of buffer and counterion (TFA) spectra. (C) Treated Raman spectrum obtained from $D_2O$ samples. The positions of the six Raman markers ($F_i$, $i=1,...,6$) are marked in red colour.

**Fig. 3** Graphic representation of the conformers embedded in solvent continuum and geometry optimized by means of M062X functional.

Conformers having (A) an extended chain, and (B) a pP-II structure. Relative energy ($\Delta E$ in kcal/mol) is reported below each conformer. Side chain orientation of each conformer is mentioned at its upper right corner. See Table S3 (Supporting Information) for conformational angles of these conformers. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white). “+” sign designates the electric charge borne by the terminal $NH_3^+$ group.

**Fig. 4** Graphic representation of the conformers embedded in solvent continuum and geometry optimized by means of M062X functional.

Conformers having (A) a helical, (B) an inverse $\gamma$-turn, and (C) a classic $\gamma$-turn structure. Relative energy ($\Delta E$ in kcal/mol) is reported below each conformer. Side chain orientation of each conformer is mentioned at its upper right corner. Intramolecular H-bonds are drawn with green broken line, of which the length (in Å) is also reported. See Table S3 (Supporting Information) for conformational angles of these conformers. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white). “+” sign designates the electric charge borne by the terminal $NH_3^+$ group.
**Fig. 5** Histograms representing the normalized Boltzmann thermal weights for the five analysed secondary structures.

Above each bar the value of the thermal weight, as expressed in percent, is reported. (A) Data obtained from the 12 conformers embedded in solvent continuum, and optimized by means of B3LYP functional. (B) Data obtained from the 12 conformers embedded in solvent continuum, and optimized by means of ωB97XD functional. (C) Data obtained from the 12 conformers embedded in solvent continuum, and optimized by means of M062X functional. (D) Data obtained from the 15 conformers interacting with 5 water molecules, all embedded in solvent continuum, and optimized by means of M062X functional.

**Fig. 6** Thermal (Boltzmann) average of the calculated Raman spectra arising from the 15 conformers of the cationic Gly-Phe-Gly embedded in a polarizable continuum, and geometry optimized by means of M062X functional.

Each Raman spectrum is multiplied by its Boltzmann weigh calculated by means of its relative energy (ΔE), of which the value (in kcal/mol) is indicated at its right-hand side. The side chain orientation (g⁺g⁻, g g⁺ or tg⁺) is reported at the left-handed side of each spectrum. Raman spectra arising from (A) extended chains, (B) helical, (C) inverse γ-turn, (D) pP-II, and (E) classic γ-turn conformers. The thermal averaged Raman spectrum obtained by summation, is displayed on the top (in green colour). The positions of the six Raman markers (Fᵢ, i=1,...,6) are marked in red colour.

**Fig. 7** Comparison between the observed and calculated Raman spectra of the cationic species of the tripeptide Gly-Phe-Gly in the middle wavenumber spectral region.

(A) Room temperature solution (H₂O) Raman spectrum. (B) Thermal averaged Raman spectrum obtained from the 12 conformers embedded in solvent continuum, and optimized by means of B3LYP functional. (C) Thermal averaged Raman spectrum obtained from the 12 conformers embedded in solvent continuum, and optimized by means of ωB97XD functional. (D) Thermal averaged Raman spectrum obtained from the 15 conformers embedded in solvent continuum, and optimized by means of M062X functional. (E) Thermal averaged Raman spectrum obtained from the 15 conformers interacting with 5 water molecules, all embedded in solvent continuum, and optimized by means of M062X functional.

**Fig. 8** Graphic representation of the conformers interacting with 5 explicit water molecules, all embedded in solvent continuum and geometry optimized by means of M062X functional.

Conformers having (A) an extended chain, and (B) a pP-II structure. Relative energy (ΔE in kcal/mol) is reported below each conformer. Side chain orientation of each conformer is mentioned at its upper right corner. See Table S4 (Supporting Information) for conformational angles. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white). “+” sign designates the electric charge borne by the terminal NH₃⁺ group.
**Fig. 9** Graphic presentations of the conformers interacting with 5 explicit water molecules, all embedded in solvent continuum and geometry optimized by means of M062X functional.

Conformers having (A) a helical, (B) an inverse γ-turn, and (C) a classic γ-turn structure. Relative energy (ΔE in kcal/mol) is reported below each conformer. Side chain orientation of each conformer is mentioned at its upper right corner. Intramolecular H-bonds are drawn with green broken line, of which the length (in Å) is also reported. See Table S4 (Supporting Information) for conformational angles. Carbon (grey), nitrogen (blue), oxygen (red), hydrogen (white). “+” sign designates the electric charge borne by the terminal NH₃⁺ group.

**Fig. 10** Thermal (Boltzmann) average of the calculated Raman spectra arising from the 15 conformers of the cationic Gly-Phe-Gly surrounded by five water molecules, all embedded in a polarizable continuum, and geometry optimized by means of M062X functional.

Each Raman spectrum is multiplied by its Boltzmann weigh calculated by means of its relative energy (ΔE), of which the value (in kcal/mol) is indicated at its right-hand side. The side chain orientation (g⁺g⁺, g⁻g⁺ or tg⁺) is reported at the left-handed side of each spectrum. Raman spectra arising from (A) extended chains, (B) helical, (C) inverse γ-turn, (D) pP-II, and (E) classic γ-turn conformers. The thermal averaged Raman spectrum obtained by summation on other spectra, is displayed on the top (in green colour). The positions of the six Raman markers (Fᵢ, i=1,...,6) are marked in red colour.
<figure><img src="image_2.png" /><figcaption>Fig.1</figcaption></figure>
<figure><img src="image_3.png" /><figcaption>Fig.</figcaption></figure>
<figure><img src="image_4.png" /><figcaption>**B**
pP-II</figcaption></figure>

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

Gly-Phe-Gly (cationic species)/continuum

A

Helix

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

B

Inverse γ-turn

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

C

Classic γ-turn

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /></figure>

<figure><img src="image_14.png" /></figure>
<figure><img src="image_15.png" /><figcaption>Fig.</figcaption></figure>
Gly-Phe-Gly (cationic species)/continuum

M062X functional

<figure><img src="image_16.png" /><figcaption>Fig.</figcaption></figure>
<figure><img src="image_17.png" /><figcaption>Fig.</figcaption></figure>
Gly-Phe-Gly (cationic species) + 5H₂O/continuum

M062X functional

<figure><img src="image_18.png" /><figcaption>B
pP-II</figcaption></figure>

Fig.

8
Gly-Phe-Gly (cationic species) + 5H₂O/continuum

M062X functional

A

Helix

<figure><img src="image_19.png" /></figure>

$\Delta E = +0.91$

<figure><img src="image_20.png" /></figure>

$\Delta E = +1.57$

<figure><img src="image_21.png" /></figure>

$\Delta E = +1.98$

B

Inverse γ-turn

<figure><img src="image_22.png" /></figure>

$\Delta E = +1.14$

<figure><img src="image_23.png" /></figure>

$\Delta E = +1.78$

<figure><img src="image_24.png" /></figure>

$\Delta E = +4.01$

C

Classic γ-turn

<figure><img src="image_25.png" /></figure>

$\Delta E = +4.37$

<figure><img src="image_26.png" /></figure>

$\Delta E = +7.66$

<figure><img src="image_27.png" /></figure>

$\Delta E = +3.38$

Fig.
Gly-Phe-Gly (cationic species) + 5H₂O/continuum

M062X functional

<figure><img src="image_28.png" /><figcaption>Fig. 10</figcaption></figure>