Determining β-sheet stability by Fourier transform infrared difference spectra

We describe here a new method for determining the conformational stability of antiparallel β‐sheets. Due to coupling between the transition dipoles, β‐sheet conformations typically exhibit a characteristic high‐frequency amide I component centered at ∼ 1680 cm−1. Using one β‐sheet protein and two small β‐hairpins, we demonstrate that this high‐frequency component, which is fairly narrow (∼ 8–10 cm−1), can be quantitatively resolved and used in thermal stability determination. Compared with the commonly used CD and fluorescence techniques, this ir method offers advantages. Since the area of this high‐frequency component is only proportional to the folded population, it eliminates the need for a priori information of the folded and unfolded baselines encountered in other methods. Thus, it is applicable to a variety of β‐sheet systems. © 2004 Wiley Periodicals, Inc. Biopolymers, 2004