In order to utilize this feature of SEIRA spectroscopy, we first have to determine the intrinsic orientation-dependence of antiparallel and parallel β-sheets. For this we constructed regular β-sheets from seven capped Ala6 strands inspired by earlier work by Keiderling and colleagues, and optimized the structures using density functional theory (DFT) at the BP86/6-31g* level of theory. We used the same level of theory in our previous work and obtained very good agreement for peak positions and relative intensities with experimental spectra. In the present case, we obtain small deviations from the experimental peak positions which are again well within the error margin of the level of theory that was used, therefore predicting experimental spectra with good accuracy. Accordingly, orientation-dependent spectra were computed using normal mode analysis for the antiparallel and parallel βsheets by rotating the structures around the y-axis indicated in the insets in Figure and, and projecting the transition dipole vectors of all amide I and amide II components onto the polarization direction, i.e., the z-axis. As shown in Figure and e, DFT-based spectra show the strongest band around 1620 -1630 cm -1 for antiparallel and parallel β-sheets, which is the collective antisymmetric C=O stretch and consistent with the most intense amide I component at 1634 cm -1 in the experimental spectra. The intensity of this mode is maximized for an orientation of close to 90° (dark blue spectra in Figure and; dark blue data points in Figure and), i.e., when the β-sheet strands are oriented horizontally. In this orientation, all C=O groups are oriented vertically. The intensity then reaches zero at a vertical orientation of 0° (yellow spectra), where C=O groups are horizontally. The second relevant amide I mode is observed in antiparallel β-sheets at 1683 cm -1 (the collective symmetric C=O stretch) and can be assigned to the 1694 cm -1 mode in experiments. This mode shows an inverted orientational behavior, i.e., it is maximized at close to 0° and minimized at roughly 85° (light blue data points in Figure ). As elaborated above, this mode is absent in parallel β-sheets. Therefore, attempting to extract the orientation dependence at 1683 cm -1 in parallel β-sheet (light blue dashed line in Figure ), we track the shoulder of other minor amide I modes (1680 -1640 cm -1 ) and obtain a similar trend as for the dark blue data points of the 1631 cm -1 mode. Finally, the amide II at ~1540 cm - 1 (green data points in Figure and) shows an approximately opposite behavior to the 1623 cm -1 in antiparallel β-sheets, which is consistent with its origin in the collective CN stretch and NH bend that oscillate perpendicular to the C=O stretches. Interestingly, this mode shows a phase shift in parallel β-sheets, i.e., with a minimum and maximum at 50° and 140°, respectively (green data points in Figure and). Using the unique orientation dependence of these three individual modes, we now have the means to model the relative intensities in the experimental spectra as a sum of an antiparallel β-sheet fraction, fap, at a specific orientation, θap, and a parallel β-sheet fraction, fpara = 1 -fap, at a specific orientation, θpara; i.e., as a function of three unknowns (see SI Section 2 for exact equations used). We note that each of the curves in Figure and f has a period of 180°, such that there are four equivalent solutions for a specific mode within a full rotation by 360° (for instance, 45°, 135°, 225° and 315° in Figure ). Therefore, combining all three modes, there can be up to four solutions for a β-sheet orientation, modelling the experimental spectra equally well with comparable relative intensities. We have projected all solutions into the β-sheet orientation range of 0° -90° and averaged the results (together with duplicates of experiments) to account for this uncertainty, which is intrinsic to our analysis.