UV Raman Demonstrates that α-Helical Polyalanine Peptides Melt to Polyproline II Conformations

Abstract

We examined the 204-nm UV Raman spectra of the peptide XAO, which was previously found by Shi et al.'s NMR study to occur in aqueous solution in a polyproline II (PPII) conformation. The UV Raman spectra of XAO are essentially identical to the spectra of small peptides such as ala(5) and to the large 21-residue predominantly Ala peptide, AP. We conclude that the non-alpha-helical conformations of these peptides are dominantly PPII. Thus, AP, which is highly alpha-helical at room temperature, melts to a PPII conformation. There is no indication of any population of intermediate disordered conformations. We continued our development of methods to relate the Ramachandran Psi-angle to the amide III band frequency. We describe a new method to estimate the Ramachandran Psi-angular distributions from amide III band line shapes measured in 204-nm UV Raman spectra. We used this method to compare the Psi-distributions in XAO, ala(5), the non-alpha-helical state of AP, and acid-denatured apomyoglobin. In addition, we estimated the Psi-angle distributions of peptide bonds which occur in non-alpha-helix and non-beta-sheet conformations in a small library of proteins.

Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

“…If the 1200-1320 cm -1 region has a significant contribution of the non-canonical amide III bands expected for Pro residues, besides the CH 2 rocking-wagging bands, the comparison between experimental bands in the amide III region and predicted amide III frequencies is good for the Asher's equation [21] (see Table 2), but not for others [22]. Despite the hydration, accurately described in the structural studies [1,6], is not included explicitly into the successful semi-empirical equation [21], the agreement between experimental and theoretical frequencies in the amide III region of (PPG) 10 is quite good. Within this assignment, the strong putative amide III bands correspond to the two proline residues in X and Y position, whereas the very minor amide III bands correpond to the Gly amide bonds.…”

“…Raman optical activity [18] and UV-Raman resonance spectroscopy [19] are also valuable tools to extract secondary structural details. Ab initio studies [20] and semi-empirical relationships [21,22] of amide I and III bands (IR inactive) with torsional angles φ and ψ are also available. The availability of polypeptides such as (PPG) 10 with a repetitive primary structure…”

“…The analysis of the 1200 and 1350 cm -1 region is problematic for Pro residues. Indeed, The analysis of these three bands in the amide III (Table 1) has been tentatively carried out by a correlation between predicting equations derived for canonical amide III bands [21,22] and the structural information of (PPG) 10 average ψ angles previously reported [1]. In fact, though the φ angles vary as well along the helix [1], the φ angular dependence of amide III frequency is supposed to be small [22].…”

“…In fact, though the φ angles vary as well along the helix [1], the φ angular dependence of amide III frequency is supposed to be small [22]. In general, predictive equations [21,22] capture the physics of the frequence dependence in that they calculate the ψ angular dependence of the coupling of the Amide III vibration with Cα-H bending. If the 1200-1320 cm -1 region has a significant contribution of the non-canonical amide III bands expected for Pro residues, besides the CH 2 rocking-wagging bands, the comparison between experimental bands in the amide III region and predicted amide III frequencies is good for the Asher's equation [21] (see Table 2), but not for others [22].…”

“…In general, predictive equations [21,22] capture the physics of the frequence dependence in that they calculate the ψ angular dependence of the coupling of the Amide III vibration with Cα-H bending. If the 1200-1320 cm -1 region has a significant contribution of the non-canonical amide III bands expected for Pro residues, besides the CH 2 rocking-wagging bands, the comparison between experimental bands in the amide III region and predicted amide III frequencies is good for the Asher's equation [21] (see Table 2), but not for others [22]. Despite the hydration, accurately described in the structural studies [1,6], is not included explicitly into the successful semi-empirical equation [21], the agreement between experimental and theoretical frequencies in the amide III region of (PPG) 10 is quite good.…”