Abstract

Kinetic IR spectroscopy was used to reveal β-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded β-sheet and an α-helix. The time-resolved IR spectra between 100 μs and 10 s were analyzed based on two consecutive intermediates, I₁ and I₂, appearing within 100 μs and with a time constant of ≈100 ms, respectively. The initial unfolded state showed broad amide I′ corresponded to a fluctuating conformation. In contrast, I₁ 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. I₂ possessed a line for solvated helix at 1,637 cm⁻¹ and major and minor lines for β-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 β-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 CO group in the β-sheet. Together with the identification of solvated helix, the core domain of I₂ was interpreted as being hydrated. Finally, slow conversion of the water-penetrated core of I₂ 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
• β-sheet