The two-dimensional IR nonlinea penta-peptide in relation to its tl

A form of two-dimensional (2D) vibrational spectroscopy, which uses two ultrafast IR laser pulses, is used to examine the structure of a cyclic penta-peptide in solution. Spectrally resolved cross peaks occur in the off-diagonal region of the 2D IR spectrum of the amide I region, analogous to those in 2D NMR spectroscopy. These cross peaks measure the coupling between the different amide groups in the structure. Their intensities and polarizations relate directly to the three-dimensional structure of the peptide. With the help of a model coupling Hamiltonian, supplemented by density functional calculations, the spectra of this penta-peptide can be regenerated from the known solution phase structure. This 2D-IR measurement, with an intrinsic time resolution of less than 1 ps, could be used in all time regimes of interest in biology. The three-dimensional (3D) structure of peptides and proteins and their fluctuations are essential properties responsible for the extremely high specificity of biological reactions. Progress in understanding biological processes such as enzyme reactions originates from the detailed knowledge of the secondary, tertiary, and quarternary structures of the participating biomolecules. Two major spectroscopic techniques are responsible for this development: x-ray scattering, which maps out the electron density of the molecule, and two-dimensional (2D)NMR spectroscopy (1-3), which can measure the distances between pairs of protons. The next step must be the determination of structures in motion over a wide range of time scales. We believe that multidimensional IR spectroscopy can address this new challenge. The IR spectra of the amide transitions provide information about secondary structural motifs of proteins and peptides. The so-called amide I band, which consists of mostly the stretching motion of the peptide backbone C=0 groups, is a strong IR absorber, which is spectrally isolated from other vibrational modes such as those from amino acid side groups. The amide I states can be viewed as vibrational excitons (4, 5) with the individual peptide groups considered as separated but coupled units. The coupling could be either through-space, such as multipole or even dipole-dipole interaction as proposed by Krimm and Bandekar (4), or through-bond, involving charge shifts and kinematic coupling via the Cr atoms of the backbone. The states, which have one excitation present in the whole assembly, can be interrogated by conventional (linear) IR absorption spectroscopy, but the information obtained is insufficient to determine the coupling Hamiltonian, from which a structure might be deduced. More information is available from nonlinear third-order spectroscopic techniques (6) such as the 2D experiment presented here. In a separated system picture both the one-exciton and two-exciton states contribute to the nonlinear IR signal in such a way that all The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. ?1734 solely to indicate this fact. PNAS is available online at www.pnas.org. 2( r spectroscopy of a cyclic iree-dimensional structure ND R BIN M. HOCHSTRASSERt? , Johnson Foundation for Research in Biophysics, University of Pennsylvania, couplings between the separate amide units are available, and in principle a 3D structure of the peptide can be deduced. The success of NMR spectroscopy as a structural tool in biology was advanced by improvements (1-3) in resolution achieved by spreading out congested spectra into a second dimension where connected groups are represented by cross peaks and can be characterized by excitation transfer. A few examples of applying similar principles to electronic and vibrational transitions have been proposed (7, 8) and realized experimentally (6, 9, 10) lately. Optical and vibrational transitions in solution dephase very rapidly compared with spin transitions so these nonlinear spectroscopies have become possible only as a result of significant recent improvements in ultrafast laser technology. The objective of such developments would be to provide alternative methods of secondary structure determination. IR spectra also can be spread into two dimensions by the variation of external conditions such as temperature or pressure (11), but these approaches differ in principle from the nonlinear spectroscopies that involve perturbations of a quasi-equilibrium distribution by an electromagnetic field. In a first attempt at nonlinear 2D IR spectroscopy two small globular peptides, scyllatoxin and apamin, (6) were examined. The results clearly manifested the coupling of the amide I states. However, even when spread into two dimensions many positive and negative bands overlapped in the 2D spectra, making it difficult to explore with confidence the coupling mechanisms. Nevertheless these experiments revealed the parameters essential for 2D spectroscopy of amide I bands, namely, the time scales of population relaxation (1.2 ps) and energy transfer between individual amide I states (>2.5 ps); the homogeneous width (10 cm-1), inhomogeneous disorder (25 cm-1), and the degree of delocalization of the amide I states (8 A). Here, we present results on a peptide that is small enough that all amide I transitions are spectrally resolved, allowing the properties of the nonlinear 2D-IR spectra and the validity of coupling models to be examined critically. The sample chosen is a cyclic penta-peptide (cyclo-Mamb-Abu-Arg-Gly-Asp), for which both the NMR and the x-ray structures are known (12). The chemical formula and the 3D structure are shown in Fig. 1. The structure of the peptide, which is stabilized by a hydrogen bond between the Mamb-l-Abu-2 peptide bond and the Arg-3-Gly-4 peptide bond, was specifically designed (12) to form a single, well-defined conformation in solution with an almost ideal type II' 3-turn centered at the Abu-Arg residues. MATERIALS AND METHODS IR Laser System. For the generation of intense IR pulses at 1,650 cm-1 [bandwidth 130 cm-1 full width at half maximum Abbreviations: 2D, two-dimensional; 3D, three-dimensional; FWHM, full width at half maximum; NMA, N-methylacetamide. tPresent address: Department of Chemistry, Pusan National University, Pusan 609-735, Korea. ?To whom reprint requests should be addressed. e-mail: hochstra@ sas.upenn.edu. '36 This content downloaded from 157.55.39.191 on Tue, 11 Oct 2016 04:40:48 UTC All use subject to http://about.jstor.org/terms Biophysics: Hamm et al. HN Gly4 Arg3 Asp5 H2N q CH3 HN OH