UV Resonance Raman-Selective Amide Vibrational Enhancement: Quantitative Methodology for Determining Protein Secondary Structure † Zhenhuan Chi, X. G. Chen, ‡ Janet S. W. Holtz, and Sanford A. Asher* Department of Chemistry, UniVersity of Pittsburgh, Pittsburgh, PennsylVania 15260 ReceiVed May 16, 1997; ReVised Manuscript ReceiVed October 29, 1997 ABSTRACT: We have directly determined the amide band resonance Raman spectra of the “average” pure R-helix, -sheet, and unordered secondary structures by exciting within the amide πfπ* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure R-helix, -sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the C R -H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media. Any fundamental understanding of protein and peptide structure, dynamics, and function requires methods to measure protein secondary structure. X-ray crystallography and two-dimensional nuclear magnetic resonance (2-D NMR) (1-4) are the most incisive protein structural probes because they can provide highly accurate three-dimensional structures. However, X-ray diffraction gives a static picture, and NMR gives only a limited amount of dynamical information (2, 4). In addition, these techniques are labor intensive and require protein samples that differ from the normally desired dilute aqueous solution phase. We report here a new methodology to determine dilute solution protein and peptide secondary structures, using UV resonance Raman spectroscopy (UVRRS) excited with a 206.5-nm CW laser directly into the amide πfπ* transitions of the peptide bonds (5-11). The resulting spectra are dominated by the amide vibrations, whose frequencies, Raman cross sections, and bandwidths depend sensitively on secondary structure. We have, for the first time, used chemometrics to directly and empirically determine the amide band resonance Raman spectra of the “average” R-helix, -sheet, and unordered secondary structures (pure secondary structure Raman spectra, PSSRS) for a series of 13 proteins. We simultaneously utilize the amide I, II, and III bands and the C R -H amide bending vibrations of these PSSRS to directly determine protein secondary structures. This new methodology is complementary to secondary structural methods, such as CD, VCD, and IR absorption spectroscopy (12-18). We show here that the ability of this UV Raman methodology to predict secondary structure is, in some cases, superior to that of CD and that the approach has significant advantages compared to CD, VCD, and IR. For example, the Raman spectra are immune from the light- scattering artifacts that plague conventional absorption measurements (17). Therefore, UVRRS can be used to examine proteins in micelles and membranes (19). We show in the accompanying paper that this UV Raman methodology can be used to study protein folding and denaturation. In this study of the acid denaturation of myoglobin, we combine the UV Raman spectra, exciting in the peptide backbone region, with spectra that result from excitation directly in the aromatic amino acid absorption bands. The amide bands characterize changes in secondary structure in the protein, such as R-helix melting, while changes in the aromatic amino acid bands suggest which helices undergo the conformational changes (20). EXPERIMENTAL SECTION Materials and Sample Preparation. Jack bean concanava- lin A (Con A), bovine pancreas trypsin (trypsin), bovine serum albumin (BSA), bovine pancreas ribonuclease A (RNase A), horse heart cytochrome c (cyt c), human hemoglobin (Hb), horse skeletal muscle myoglobin (Mb), bovine pancreas R-chymomyosin (Chy), porcine pancreas elastase (Elas), chicken egg white lysozyme (Lyso), human erythrocyte carbonic anhydrase (Carb), porcine muscle triosephosphate isomerase (Trio), bovine pancreas insulin (Ins), tyrosine (Tyr), phenylalanine (Phe), tryptophan (Trp), histidine (His), arginine (Arg), cysteine (Cys), proline (Pro), methionine (Met), cystine, and the bromide salt of poly(L- lysine) (PLL, MW ) 22 000) and the sodium salt of poly- (L-glutamic acid) (PGA, MW ) 26 500) were purchased from Sigma Chemical Co. (St. Louis, MO). Sodium per- † We gratefully acknowledge support from NIH Grant R01GM30741- 15. * To whom correspondence should be addressed. Phone: (412) 624- 8570. Fax: (412) 624-0588. E-mail: asher+@pitt.edu. ‡ Present address: Section on Metabolic Analysis and Mass Spec- trometry, NICHD, National Institute of Health, 10 Center Dr., MSC 1580, Room 6C208, Bethesda, MD 20892. 2854 Biochemistry 1998, 37, 2854-2864 S0006-2960(97)01160-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 02/11/1998