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Kinetic IR spectroscopy was used to reveal ␤-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded ␤-sheet and an ␣-helix. The timeresolved IR spectra between 100 s and 10 s were analyzed based on two consecutive intermediates, I 1 and I2, appearing within 100 s and with a time constant of Ϸ100 ms, respectively. The initial unfolded state showed broad amide I corresponded to a fluctuating conformation. In contrast, I 1 possessed a feature at 1,636 cm ؊1 for solvated helix and weak features assignable to turns, demonstrating the rapid formation of helix and turns. I 2 possessed a line for solvated helix at 1,637 cm ؊1 and major and minor lines for ␤-sheet at 1,625 and 1,680 cm ؊1 , respectively. The splitting of the major and minor lines is smaller than that of the native state, implying an incomplete formation of the ␤-sheet. Furthermore, both major and minor lines demonstrated a low-frequency shift compared to those of the native state, which was interpreted to be caused by hydration of the CAO group in the ␤-sheet. Together with the identification of solvated helix, the core domain of I 2 was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I 2 to the dehydrated core of the native state was observed. We propose that both the expulsion of water, hydrogen-bonded to main-chain amides, and the completion of the secondary structure formation contribute to the energetic barrier of the rate-limiting step in SMN folding.protein folding dynamics ͉ ␤-sheet

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