Dehydration of main-chain amides in the final folding step of single-chain monellin revealed by time-resolved infrared spectroscopy

Tetsunari Kimura; Akio Maeda; Shingo Nishiguchi; Koichiro Ishimori; Isao Morishima; Takashi Konno; Yuji Goto; Satoshi Takahashi

doi:10.1073/pnas.0801316105

New Research In

Edited by Robert L. Baldwin, Stanford University Medical Center, Stanford, CA, and approved July 10, 2008 (received for review February 10, 2008)

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.

Footnotes

**To whom correspondence should be addressed. E-mail: st{at}protein.osaka-u.ac.jp
Author contributions: T. Kimura, K.I., I.M., Y.G., and S.T. designed research; T. Kimura, A.M., and K.I. performed research; S.N. and T. Konno contributed new reagents/analytic tools; T. Kimura and S.T. analyzed data; and T. Kimura and S.T. wrote the paper.
↵^‡Present address: Beckman Institute, California Institute of Technology, Pasadena, CA 91125.
↵^§Present address: Division of Chemistry, Graduate School of Science, Hokkaido University, Sapporo 060-0810, Japan.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
This article contains supporting information online at www.pnas.org/cgi/content/full/0801316105/DCSupplemental.
© 2008 by The National Academy of Sciences of the USA