JOURNAL OF RAMAN SPECTROSCOPY J. Raman Spectrosc. 2006; 37: 248–254 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jrs.1455 The conformations adopted by the octamer peptide (AAKA) 2 in aqueous solution probed by FTIR and polarized Raman spectroscopy † Thomas Measey and Reinhard Schweitzer-Stenner ∗ Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104, USA Received 27 April 2005; Accepted 19 July 2005 In an effort to further elucidate the chain length dependence of the poly-L-proline 3 1 helical structure (PPII) in short alanine-based polypeptides, we utilize FTIR, isotropic and anisotropic Raman, and electronic circular dichroism (ECD) spectroscopies to probe the amide I ′ band of the unblocked octamer H-Ala-Ala- Lys-Ala-Ala-Ala-Lys-Ala-OD in D 2 O. Our results are in disagreement with recent notions suggesting that the 3 1 helical structure increases with an increase in the number of alanine residues. 1,2 Simulations of the spectra were carried out and found to best reproduce experimental spectra using parameters that suggest a 40/60% PPII/b-strand (ß s ) mixture. The amount of PPII in the octamer is found to be significantly lower than what was determined for unblocked alanine-based peptides of comparable or even larger size. This is confirmed by the relatively weak ECD minimum and maximum at ∼195 and ∼220 nm, respectively, for the octamer relative to tri- and tetraalanine. 3 In this paper we also address a recent issue concerning the validity of the delocalized character of the amide I mode in unfolded polypeptides containing a significant amount of PPII structure. We conclude that experimental evidence strongly supports the vibrational coupling model used to interpret the amide I mode in unfolded polypeptides. Copyright  2006 John Wiley & Sons, Ltd. KEYWORDS: polyproline II; polarized Raman spectroscopy; FTIR spectroscopy; alanine-based peptides INTRODUCTION Over the last 15 years, alanine-based peptides have become a valuable tool to study helix , ‘coil’ transitions in the absence of nonlocal forces imposed by the tertiary structure in (globular) proteins. 4 Chakrabartty et al. 5 have carried out numerous studies to identify multiple contributions to the stabilization of ˛ R . The significance of intrinsic propensities obtained by analyzing the thermal unfolding of 58 alanine- based peptides containing ‘guest residues’ at specific central and terminal positions has been a matter of a controversial debate. The authors reported a propensity scheme clearly dominated by alanine. Besides alanine, only protonated † Presented as part of a commemorative issue for Wolfgang Kiefer on the occasion of his 65th birthday. L Correspondence to: Reinhard Schweitzer-Stenner, Department of Chemistry, Drexel University, 3141 Chestnut Street, Philadelphia, PA 19104. E-mail: RSchweitzer-Stenner@drexel.edu Contract/grant sponsor: National Science Foundation; Contract/grant number: MCB-0318749. Contract/grant sponsor: ACS-Petroleum Research Funds; Contract/grant number: PRF # 37406-AC. arginine was found 6 to exhibit some preference for ˛ R . These results have been heavily debated in the literature. 7–11 Many of the folding/unfolding studies on alanine-based peptides are based on the assumption that the corresponding unfolded state is a random coil. 12 – 14 In a random coil, the energy difference among sterically accessible backbone con- formers are on the order of kT (k: Boltzmann constant, T: abso- lute temperature). The energy landscape appears essentially featureless, and a Boltzmann-weighted ensemble of such polymers would populate this landscape uniformly. 1,14 – 16 However, more recent experimental and theoretical studies on small, nonhelix-forming peptides challenge this notion. Shi et al. 17 combined NMR with electronic circular dichroism (ECD) experiments to investigate the structure of the 11-mer Ac-XX-A 7 -Ooamide (XAO peptide, X: diaminobutyric acid, O: ormithine) at different temperatures between 2 and 55 ° C. Such a peptide was generally believed to be structurally disordered. However, the authors’ results indicate that it predominantly adopts a PPII conformation at room temper- ature. At high temperature, they observed an admixture of an extended, ˇ-sheet like conformation (ˇ s ). Both observa- tions suggest that the coil state of this peptide can be ascribed Copyright  2006 John Wiley & Sons, Ltd.