Characteristic Raman lines of phenylalanine analyzed by a multiconformational approach

Abstract

Thanks to a considerable modulation of electronic polarizability, six phenylalanine (Phe) vibration modes located at ca. 1606, 1586, 1207, 1031, 1004 and 622 cm−1 appear as intense or medium bands in the Raman spectra of peptides and proteins, as confirmed by the Raman data collected from free amino acid, somatostatin and bovine serum albumin (BSA). To get information on the nature and location of these lines, we resorted to a multiconformational analysis which consists in a systematic investigation of the structural and vibrational features of hydrated Phe in a conformational space depending on four angular variables: φ, ψ, χ1 and χ2. The first two variables correspond to the Phe backbone torsion angles, whereas the latter two refer to its side chain. Based on a protocol described in an accompanying report on glycine and its protonated and deprotonated species, we have prepared an initial set of 123 initial clusters of Phe + 5H2O, including all plausible values of the above mentioned conformational angles. The results of their geometry optimization, by means of the density functional theory using the B3LYP hybrid functional, were first analyzed through the comparison between the E(χ1, χ2) energy maps obtained either by an explicit or by an implicit hydration model. Then, a set of nine doubly minimized clusters corresponding to the deepest local minima were used for further structural and vibrational analysis. Beyond providing a reliable assignment for the above mentioned characteristic Raman lines, the theoretical spectrum allowed us to carry out an overview of the whole observed data of Phe in aqueous solution. Copyright © 2013 John Wiley & Sons, Ltd.

All characteristic Raman markers of tyrosine and tyrosinate originate from phenol ring fundamental vibrations

“…This level of theory has provided reliable theoretical data for other amino acids. [11,41] To take into account the hydration effects, two different models were considered. The first one is a purely implicit approach, by placing the solute in a cavity within the solvent reaction field.…”

“…[11] Briefly, a systematic search on the most plausible conformers of the zwitterionic free phenylalanine, Phe(0) (Scheme 1), as a function of the four angular variables (φ, ψ, χ 1 , and χ 2 ), where (φ, ψ) and (χ 1 , χ 2 ) refer to the backbone and side chain torsion angles, respectively, had made possible the construction of 123 independent conformers of Phe(0) + 5H 2 O. [11] The five water molecules involved in each cluster have allowed achievement of a reasonable hydration state of the two charged end groups (NH 3 + and COO À ) of the amino acid backbone. Geometry optimization of these conformers had revealed nine clusters, of which the relative energy (ΔE) was located in an interval of 1.09 kcal mol À1 .…”

“…Calculated Raman spectrum derived from these low-energy clusters had shown its ability to assign the observed room temperature solution Raman spectrum of phenylalanine at pH~7. [11] Here, by adding a hydroxyl group to the phenyl ring, we have first constructed nine initial clusters of Tyr(0) + 5H 2 O. In a second step, to examine the influence of hydration on the phenol ring hydroxyl group, two other series of initial clusters were constructed, referred to as Tyr(0) + 6H 2 O and Tyr(0) + 7H 2 O, respectively.…”

“…In the former clusters, the phenol hydroxyl interacts with only one water molecule, whereas in the latter ones, two water molecules interact with the same molecular group. As in the case of glycine [41] and phenylalanine, [11] the final Figure 1. Comparison of the observed Raman spectra in aqueous solution of zwitterionic Tyr (bottom), the tripeptide Gly-Tyr-Gly (middle), and lysozyme (top) in the 1750-600-cm À1 spectral region.…”

“…Because of their high statistical weights, only those clusters with a relative energy (ΔE) not larger than~1.2 kcal mol À1 can significantly contribute to the final calculated spectrum at room temperature. [11,41] The thermal average of the calculated Raman spectra obtained from the different Figure 4. Comparison between the theoretical Raman spectra obtained from p-cresol and zwitterionic Tyr in the 900-800-cm À1 spectral region.…”