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Orientation of Peptides in Aqueous Monolayer Films. Infrared Reflection-Absorption Spectroscopy Studies of a Synthetic Amphipathic β-Sheet Zhi Xu, Joseph W. Brauner, Carol R. Flach, and Richard Mendelsohn* Department of Chemistry, Newark College of Arts and Science, Rutgers University, 73 Warren Street, Newark, New Jersey 07102 Received December 5, 2003. In Final Form: February 19, 2004 Infrared reflection-absorption spectroscopy (IRRAS) intensities of the Amide I vibration are used to develop a quantitative approach for determining the Euler angles that describe the orientation of protein β-sheets in aqueous monolayer films. A synthetic amphipathic peptide, Val-Glu-Val-Orn-Val-Glu-ValOrn-Val-Glu-Val-Orn-Val-OH is used as a test case. The pattern of Amide I frequencies suggests that the molecule is organized as an antiparallel β-sheet at the air/water interface. The model used to simulate the Amide I intensities reveals that the β-sheet has a slight preferential alignment parallel to the direction of compression; i.e., deviation from uniaxial symmetry is observed. In addition, the sheet is found to lie flat on the aqueous surface, with (presumably) the polar side chains interacting with the aqueous subphase. Limitations and advantages of the theoretical approach are discussed.
Introduction Infrared reflection-absorption spectroscopy (IRRAS), developed by Dluhy and co-workers in the mid-1980s,1,2 provides a unique means of acquiring molecular structure information from lipid/protein monolayer (Langmuir) films in situ at the air/water interface. Since the initial experiments on Langmuir films of lipids, IRRAS technology has improved to the point where good quality spectra of proteins, alone or mixed with lipids in monolayers, may be acquired.3-5 Two types of spectral information result from IRRAS measurements, frequencies and intensities. Frequencies are easier to interpret, and they provide information in the usual way about molecular structure and interactions. The most useful information about protein structure comes from Amide I frequencies (mostly peptide bond CdO stretch) which are well correlated with protein secondary structures. The second, less widely used aspect of IRRAS involves the determination of orientation from quantitative evaluation of measured band intensities. Several IRRAS studies have determined acyl chain orientation (as a mean tilt from the surface normal) in ordered phospholipid5 and fatty acid monolayer films.6 In general, the observed chain tilts are consistent (to within a degree or two) with those derived from X-ray scattering measurements. Recently, the Rutgers group has used an improved IRRAS apparatus to show that different acyl chain tilt angles occur in each chain of a fatty acid homogeneous derivative of ceramide 2.7 In addition, a novel method has been reported for functional group orientation in lipid molecules, namely, the CdO orientation in behenic * To whom correspondence should be addressed. E-mail:
[email protected]. (1) Dluhy, R. A.; Cornell, D. G. J. Phys. Chem. 1985, 89, 3195. (2) Dluhy, R. A. J. Phys. Chem. 1986, 90, 1373. (3) Mendelsohn, R.; Brauner, J. W.; Gericke, A. Annu. Rev. Phys. Chem. 1995, 46, 305. (4) Ulrich, W.-P.; Vogel, H. Biophys. J. 1999, 76, 1639. (5) Bi, X.; Taneva, S.; Keough, K. M. W.; Mendelsohn, R.; Flach, C. R. Biochemistry 2001, 40, 13659. (6) Blaudez, D.; Buffeteau, T.; Castaings, N.; Desbat, B.; Turlet, J. M. J. Chem. Phys. 1996, 104, 9983. (7) Flach, C. R.; Xu, Z.; Bi, X.; Brauner, J. W.; Mendelsohn, R. Appl. Spectrosc. 2001, 55, 1060.
acid methyl ester and for both carbonyls in sn2-13C 1,2distearoylphosphatidylcholine.8 A few IRRAS studies have also reported the orientation of helical proteins in mixed Langmuir films with phospholipids. For example, equations were derived to determine the orientation of the helical pulmonary surfactant protein SP-C in monolayers of 1,2-dipalmitoylphosphatidycholine (DPPC). At a surface pressure of ∼30 mN/m, the helix was shown to be tilted at ∼70° from the surface normal.9 A polarization-modulation (PM) IRRAS study reported qualitative pressure-dependent orientation for gramicidin A in mixed lipid-peptide monolayers.4 Determination of the orientation of β-sheet-containing peptides is an important extension of IRRAS measurements since reports of β-forms, e.g. β-sheets in amyloidrelated diseases and in defensin-like antimicrobial peptides, have become prevalent. A qualitative evaluation of antiparallel β-sheet orientation can be obtained from the sign (positive or negative) of Amide I band components observed using p-polarized radiation or PM-IRRAS measurements. PM-IRRAS was used in a previous study where a model amphipathic β-sheet peptide was found to lie approximately flat at the air/water interface.10,11 The technical problems in the quantitative analysis of IRRAS data for β-sheet orientation are addressed in the current work. The method is an extension of the formalism developed in this laboratory for the orientation of functional groups.8 The theoretical formalism is tested with an amphipathic β-sheet peptide where the chemical structure of the peptide is assumed to cause the plane of the sheet to lie parallel to the aqueous surface. Experimental Procedures Materials. Chloroform, methanol, EDTA, and HPLC grade water were obtained from Fisher Scientific (Pittsburgh, PA). (8) Brauner, J. W.; Flach, C. R.; Xu, Z.; Bi, X.; Lewis, R. N. A. H.; McElhaney, R. N.; Gericke, A.; Mendelsohn, R. J. Phys. Chem. B 2003, 107, 7202. (9) Gericke, A.; Flach, C. R.; Mendelsohn, R. Biophys. J. 1997, 73, 492. (10) Castano, S.; Desbat, B.; Dufourcq, J. Biochim. Biophys. Acta 2000, 1463, 65. (11) Buffeteau, T.; Le Calvez, E.; Castano, S.; Desbat, B.; Blaudez, D.; Dufourcq, E. J. J. Phys. Chem. B 2000, 104, 4537.
10.1021/la0304316 CCC: $27.50 © 2004 American Chemical Society Published on Web 04/02/2004
Peptide Orientation in Monolayer Films Trizma (tris[hydroxymethyl]aminomethane) hydrochloride and sodium chloride were purchased from Sigma (St. Louis, MO). D2O with 99.9% isotopic enrichment was purchased from Cambridge Isotope Laboratories (Andover, MA). A peptide designed to be an amphipathic β-sheet (termed β-13) was a gift from Professor John Taylor, Rutgers University, and was originally received from the Kaiser laboratory.12 Its purity was verified by mass spectrometry. The peptide sequence is ValGlu-Val-Orn-Val-Glu-Val-Orn-Val-Glu-Val-Orn-Val-OH. The Orn residues are Nδ-trifluoroacetyl-L-ornithine. IRRAS/Langmuir Trough Accessory. The IRRAS equipment has been described in detail previously.7 Briefly, spectra were acquired with a Bruker Instruments Equinox 55 spectrometer equipped with an external variable angle reflectance accessory, the XA511. The accessory is a self-contained computercontrolled unit attached to the external port on the spectrometer coupled to a custom-designed Langmuir trough with a Nima model PS4 surface pressure sensor (Nima Technology Ltd., Coventry, England). The IR beam is directed through the external port in the spectrometer and is reflected by three mirrors in a rigid mount prior to being focused on the water surface. Computerdriven stepper motors rotate the mirrors to obtain the desired angle of incidence. A wire-grid polarizer is placed into the optical path just prior to the point where the beam impinges on the water surface. The efficiency of polarizer, approximately 99.2%, was previously determined.7 The reflected light is collected at the same angle as the angle of incidence, follows an equivalent mirror path, and is directed onto a narrow band mercury cadmium telluride detector. The entire experimental setup is enclosed and purged to keep the relative humidity levels both low and as constant as feasible. A sample shuttle, driven by a computer-controlled stepper motor, allows interferograms from the reference side (aqueous surface) and from the sample side (film-covered surface) of the trough to be collected in alternating fashion. The number and size of coadded interferogram blocks can be varied to provide a high signalto-noise ratio and to most efficiently compensate for water vapor. IRRAS Sample Preparation and Spectral Acquisition. The β-13 peptide was dissolved in 150 mM NaCl D2O solution at ∼0.5 mg/mL. A D2O-based subphase consisting of 150 mM NaCl, 0.1 mM EDTA in 5 mM Tris buffer, pD 6.9, was used for all experiments. This subphase is used to eliminate the reflectance-absorbance (RA) from the H2O bending vibration and to lessen the absorbance from the rotation-vibration bands of water vapor, both of which occur in the conformation-sensitive Amide I region of proteins. The subphase temperature was controlled at 20.0 ( 0.5 °C. Peptide monolayers were formed by spreading 20 µL of the β-13 peptide solution on a clean surface (maximum surface area of 86 cm2). Initial surface pressure values were 0 mN/m and 30 min was allowed for film relaxation/equilibration. Films were continuously compressed at a speed of 0.02-0.03 nm2/(lipid‚molecule‚min), and surface pressure-molecular area (π-A) isotherms were recorded. A relaxation period of at least 10 min was allowed between stopping the barrier at the desired surface pressure values and IRRAS spectral acquisition. IRRAS experiments were repeated a minimum of four times. Interferograms were collected with the use of sample shuttling to compensate for the residual water vapor rotation-vibration bands in the Amide I region. A total of 2048 scans were acquired at ∼8 cm-1 resolution, in four blocks of 512 scans each, co-added, apodized with a Blackman-Harris three-term function, and fast Fourier transformed with one level of zero-filling to produce spectral data encoded at ∼4 cm-1 intervals. Spectra were acquired over the desired range of incident angles using p-polarized light followed by data collection using s-polarized light. IRRAS Data Analysis. IRRAS spectra were baseline corrected using Grams/32 software (Galactic Industries). Peak positions were determined using a center of gravity algorithm provided by the National Research Council of Canada. Intensities were determined as peak heights.
Theoretical Considerations Orientations. A local right-handed Cartesian coordinate system (depicted in Figure 1), abc, is attached to the (12) Osterman, G.; Kaiser, E. T. J. Cell. Biochem. 1985, 29, 57.
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Figure 1. Coordinate systems for the IRRAS simulations. Top left: A local right-handed Cartesian coordinate system, abc, is attached to the β strand such that the “c” axis is in the direction of the strand, the “a” axis is in the plane of the sheet perpendicular to the “c” axis and the “b” axis is perpendicular to both “a” and “c”. The orientation of the transition moment vector, M, associated with the Amide I band in the local system is described by a polar angle R and an azimuthal angle β. R is the angle between the transition moment and the “c” axis and β is the angle of the projection of the transition moment in the “ab” plane from the “a” axis. Center: A laboratory Cartesian coordinate system is selected such that the positive Z axis is along the surface normal, the X axis is in the direction of the barrier movement in the Langmuir trough, and the YZ plane is the plane of incidence. The orientation of the local system in the laboratory frame is described by the Euler angles, θ, φ, and χ. θ and φ are the ordinary polar coordinates of the “c” axis in the XYZ system. θ is the angle from OZ to Oc, where O is the origin, φ is the angle in the XY plane from OX to the projection of Oc on the XY plane, and χ is an angle in the “ab” plane measuring the rotation clockwise about the “c” axis (from the Z axis).
β strand such that the “c” axis is in the direction of the strand, the “a” axis is in the plane of the sheet perpendicular to the “c” axis, and the “b” axis is perpendicular to both “a” and “c”. The orientation of the transition moment vector, M, associated with the Amide I band in the local system is described by a polar angle R and an azimuthal angle β. R is the angle between the transition moment and the “c” axis and β is the angle of the projection of the transition moment in the “ab” plane from the “a” axis. A laboratory right-handed Cartesian coordinate system is selected such that the positive Z axis is along the surface normal, the X axis is in the direction of the barrier movement in the Langmuir trough, and the YZ plane is the plane of incidence. The orientation of the local system in the laboratory frame (Figure 1) is described by the Euler angles, θ, φ, χ, as defined in Appendix I of Wilson, Decius, and Cross.13 θ and φ are the ordinary polar coordinates of the “c” axis in the XYZ system. θ is the angle from OZ to Oc, where O is the origin, φ is the angle in the XY plane from OX to the projection of Oc on the XY plane, and χ is an angle in the “ab” plane measuring the rotation clockwise about the “c” axis (from the Z axis). The components of M in the local system are given by, ma ) M sin R cos β, mb ) M sin R sin β, and mc ) M cos R. The magnitude of M depends on the oscillator strength of the Amide I band. In the case of the β-sheet peptide, the geometry requires that R ) 90° and β ) 0°, so that ma ) M, mb ) 0, and mc ) 0. Therefore, the Amide I transition moment lies mostly along the local axis “a”. In this (13) Wilson, E. B.; Decius, J. C.; Cross, P. C. Molecular Vibrations. The Theory of Infrared and Raman Vibrational Spectra; McGraw-Hill: New York, 1955.
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instance, the equations of Wilson, Decius, and Cross13 lead to the fact that the extinction coefficients (kx,, ky, and kz) in the X, Y, and Z (laboratory fixed system) directions have components
kx ) (M cos θ cos φ cos χ - M sin φ sin χ)2 ky ) (M cos θ sin φ cos χ + M cos φ sin χ)2 kz ) (-M sin θ cos χ)2 Reflectance Equations. Polarized IRRAS spectra of the Amide I mode of β-13 on D2O have been simulated with the reflectance equations from the optical theory of Kuzmin and his associates14,15 which are applied to a thin layer (film) between two semi-infinite phases. The orientation of the β-sheet requires determination of the three Euler angles, θ, φ, and χ, as mentioned above. The theory requires knowledge of the optical constants (index of refraction, n, and extinction coefficient, k) of all three layers as well as the film thickness. We have discussed the use of these equations several times in previous studies.7,8 The first semi-infinite phase is air which is isotropic, practically nonabsorbing, and has an index of refraction of essentially 1. The second semi-infinite phase is D2O which is also taken to be isotropic and whose indices of refraction and extinction coefficients in the 1600-1750 cm-1 region are taken from Bertie’s data.16 The film was taken to be anisotropic with distinct values for the three Cartesian components of its optical constants in the laboratory frame as noted above. Results Typical s- and p-polarized IRRAS spectra of the Amide I stretching region (1600-1850 cm-1) for the β-13 peptide on D2O, acquired at a surface pressure of 3 mN/m at several angles of incidence, are shown in parts A and B of Figure 2, respectively. The intense band at 1618 cm-1 coupled with the presence of weak features in the 1685-1710 cm-1 region reveals the presence of an antiparallel β-sheet structure.17 Only the intense mode at 1618 cm-1 is analyzed in the current work. The qualitative variation of the intensity for this mode as a function of angle of incidence in each polarization is consistent with the transition moment lying primarily in the laboratory X, Y plane, i.e., with the sheet lying preferentially parallel to the monolayer surface. The change in sign in the ppolarized component occurs near the Brewster angle (∼53°) for D2O. In earlier calculations of β-sheet orientation, uniaxial symmetry about the Z axis was assumed.10,11 This is equivalent to assuming φ ) 45°. A recent study from this laboratory,8 which took advantage of the high quality of spectra available, shows that this assumption may not be exactly correct. For example, for behenic acid methyl ester, φ was found to be 46.5° indicating that the plane of the zigzag carbon backbone has a slight preferential alignment in the Y direction, i.e., perpendicular to the direction of compression. (14) Kuzmin, V. L.; Michailov, A. V. Opt. Spectrosc. (USSR) 1981, 51, 383. (15) Kuzmin, V. L.; Romanov, V. P.; Michailov, A. V. Opt. Spectrosc. 1992, 73, 1. (16) Bertie, J. E.; Ahmed, M. K.; Eysel, H. H. J. Phys. Chem. 1989, 93, 2210. (17) Surewicz, W. K.; Mantsch, H. H.; Chapman, D. Biochemistry 1993, 32, 389.
Figure 2. Typical IRRAS spectra of the Amide I stretching region (1500-1750 cm-1) for the β-13 peptide on a D2O subphase acquired at a surface pressure of 3 mN/m at several angles of incidence using (A) s-polarized radiation and (B) p-polarized radiation.
To explore possible variation in φ, initial simulations assumed that θ ) χ ) 90°. This is physically the most reasonable model for peptide orientation (strand flat on the surface, polar groups oriented toward the aqueous surface). To determine the deviation from uniaxial symmetry, we have utilized the dichroic (p-polarized/spolarized) intensity ratio for comparison of experiment with theory. The advantage of this approach,8 widely used in attenuated total reflectance (ATR) spectroscopy, is that it eliminates the need for independent determination of the extinction coefficient. A comparison of the experimental with theoretical values of the intensity ratio for various assumed values of φ, at two different monolayer surface pressures, is given in Figure 3. In this instance the best values of φ are seen to be ∼43.5° and 42°, for surface pressures of 3 and 18 mN/m, respectively. This value indicates that the strand has a slight preferential alignment in the “X” direction, i.e., parallel to the compression direction. Results from more elaborate simulations where both φ and χ were allowed to vary are shown in Figure 4 for the peptide monolayer at a surface pressure of 3 mN/m. The minimum difference (sum of squares) between the experimental and calculated curves clearly occurs near φ ) 43.5° and χ ) 90°. The best value of χ ranges from ∼83 to 90° while φ is well-determined with a precision of ∼0.5°. Discussion The current model extends that previously developed for determination of CdO orientation in monolayers of esters and phospholipids. The extension to β-sheet structure requires addition of a variable, χ, which measures
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Figure 4. Results from simulations where θ ) 90° and both φ and χ were allowed to vary for the peptide monolayer at a surface pressure of 3 mN/m. The minimum difference (sum of squares) between the experimental and calculated curves clearly occurs near φ ) 43.5° and χ ) 90°. The best value of χ ranges from ∼83 to 90° while φ is well determined with a precision of ∼0.5°.
Figure 3. Comparison of the experimental with theoretical values of the IRRAS p/s intensity ratio for various assumed values of φ, at (A) a monolayer surface pressure of 3 mN/m and (B) a monolayer surface pressure of 18 mN/m. In this instance the best values of φ are seen to be ∼43.5° and 42°, for surface pressures of 3 and 18 mN/m, respectively. Values 70° yield comparable minimal differences between experimental and simulated results with χ ) 90° and φ ) 42.5°. In contrast, the sensitivity to φ is very high (see Figure 3), and slight deviation from cylindrical symmetry may readily be determined for particular values of χ and θ. Complementary considerations apply for different values of χ and θ. For example, in the extreme and unlikely case where the β-sheet stands on edge (χ ) 0° and θ ∼90°, CdO bonds pointing in the Z direction), then φ cannot be determined at all. Thus the sensitivity of these experimentally derivable parameters to variations in orientation depends on their actual values. The utility of the overall theoretical formalism is demonstrated here. Some improvements to the current approach may be envisioned. The current IRRAS experiments offer only the possibility of precise measurements of the ν⊥(π, 0)
mode (1618 cm-1) in which the transition moment direction is in the plane of the sheet normal to the chain axis, i.e., along the CdO bond direction. Should improved data become available for other vibrations with transition moment directions perpendicular to the CdO bond, then the complete determination of β-sheet orientation, independent of the actual values of χ, θ, and φ, could be calculated. As a possible candidate mode, the IR active parallel polarized ν|(0, π) mode at ∼1685 cm-1 of the Amide I contour is suggested. This band is currently too weak in the IRRAS spectra (see Figure 2) for quantitative measurements. Whereas a few IRRAS experiments have successfully determined the orientation of R-helices in Langmuir films,4,9 the quantitative orientation of β-sheets on aqueous surfaces has not been extensively addressed. Marsh has discussed the general case for the angular dependences of dichroic ratios of the Amide bands for β-sheets in lipid bilayers or multilayers using ATR spectroscopy.18 He notes, as suggested in the current case, that the orientation of the β-sheet structure requires the dichroic ratio for two bands that have transition moments oriented parallel and perpendicular to the strand axis. Finally, there are several biologically important problems where the determination of β-sheet structure and orientation in lipid environments is of interest. These include integral membrane proteins such as the acetylcholine receptor, porins, and some membrane ATPases. Changes in the protein orientation in response to external perturbations are not conveniently monitored by crystallography and may be followed by extensions of the current approach. Acknowledgment. This work was supported by the US Public Health Service through Grant GM 29864 to R.M. We thank Professor John Taylor for his gift of the peptide. LA0304316 (18) Marsh, D. Biophys. J. 1997, 72, 2710.
