Structure Analysis of Dipeptides in Water by Exploring and Utilizing the Structural Sensitivity of Amide III by Polarized Visible Raman, FTIR-Spectroscopy and DFT Based Normal Coordinate Analysis Reinhard Schweitzer-Stenner,* ,² Fatma Eker, ‡ Qing Huang, ² and Kai Griebenow ² Department of Chemistry and Biology, UniVersity of Puerto Rico, Rı ´o Piedras Campus, P.O. Box 23346, San Juan, Puerto Rico 00931-3346 Piotr A. Mroz and Pawel M. Kozlowski Department of Chemistry, UniVersity of LouisVille, LouisVille, Kentucky 40292 ReceiVed: October 4, 2001; In Final Form: January 17, 2002 A series of dipeptides AX and XA (X ) G, K, L, S, and V) were investigated by polarized visible Raman and FTIR-spectroscopy to examine the conformational determinants of the amide III band. A spectral decomposition combined with density functional calculations revealed that the amide III band has a multicomponent structure in that three different modes contribute to amide III vibrations. One of them (amide III 2 ) dominates the Raman spectra particularly of the cationic species. Its normal mode displays an in-phase combination of NH and C R1 H in plane bending vibrations, which makes it sensitive to changes of the dihedral angle ψ. Indeed, our Raman data show that amide III 2 varies with ψ but remains practically unaffected by variation of φ in the region between -95° and -75°, which is sampled by the investigated AX peptides. Our data support the Lord hypothesis that amide III depends solely on ψ (Lord, R. Appl. Spectrosc. 1977, 31, 187) but specifies to which of the amide III modes this statement applies. Our data further reveal that all amide III modes can interact with side chains vibrations. For some residues this causes a mode delocalization which yields a reduction of the Raman cross section. Amide S, which is a structure sensitive band resonance enhanced with UV-excitation, disappears for ψ-values outside of the -sheet region due to changes of the normal mode compositions of several modes between 1300 and 1420 cm -1 . This explains its absence in the UV-Raman spectra of R-helical structures. Our data suggest that all AX peptides exhibit ψ angles around 150°. Introduction Twenty-four years ago, Lord published his classical paper 3 about “Strategy and Tactics in the Raman Spectroscopy of Biomolecules”, in which he postulated that the frequency of the amide III band in the Raman spectra of peptides and proteins is a well-defined function of the dihedral angle ψ (NC R CN torsion). If this hypothesis is verified, Raman spectroscopy can be used to determine the distribution of ψ angles among the peptides linkages of a protein. Despite its importance for the structure analysis of peptides and proteins in solution significant efforts to check Lord’s hypothesis have been undertaken only recently. Williams and co-workers 2 measured the amide III band region in the Raman spectra of a series of AX and XA dipeptides (X labels various amino acid residues). There results lead them to suggest that the amide III frequency depends on the dihedral angle φ rather than on ψ, in contrast to Lord’s proposal. Jordan and Spiro 3 investigated various secondary amide derivatives by UV reso- nance-Raman spectroscopy and found evidence that the bending vibration of the C R H bond adjacent to the carbonyl group is admixed to amide III. They suggested this vibrational mixing as a possible physical source for the ψ-dependence of amide III proposed by Lord. 1 Ianoul et al. 4 found that the amide III band position in the UV-Raman spectra of a series of acetylated amino acid esters with different side chain derivatives decreases by 7 cm -1 as the preferred φ angle decreases from -75 to -95°. 5 This experimental result was reproduced by DFT based normal mode calculations for minimized structures with different constrained φ-angles. Moreover, the authors carried out similar calculations for (blocked) alanylalanine and found a similar though significantly reduced φ-dependence of amide III. In a most recent study, Asher et al. 6 investigated in more detail the conformational sensitivity of amide III for alanylalanine, its isotopic derivatives and some acetylated amino acids and found that the NH in-plane bending mode of the peptide group and the respective (C)C R H bending mode are coupled. The eigen- vector of amide III exhibits an in-phase combination of (C)C R H and NH in plane bending, in agreement with earlier suggestions by Jordan and Spiro. 3 Results of normal mode calculations for minimized structures with different constrained ψ-angles suggest that the coupling between the two bending modes exhibits a sinusoidal dependence on ψ and a linear dependence on the distance between the hydrogen atoms of (C)C R H and NH. The above vibrational mixing is maximal for ψ ≈ 120°, which corresponds to the -sheet region of the Ramachandran plot and minimal for right-handed R-helical conformations where * To whom correspondence should be addressed. Phone: (787) 764- 0000 (2417). Fax: (787) 756-8242. E-mail: rstenner_upr_chemistry@gmx.net. ² Department of Chemistry, University of Puerto Rico. ‡ Department of Biology, University of Puerto Rico. 4294 J. Phys. Chem. B 2002, 106, 4294-4304 10.1021/jp0137118 CCC: $22.00 © 2002 American Chemical Society Published on Web 03/30/2002