期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2008
卷号：105
期号：36
页码：13391-13396
DOI：10.1073/pnas.0801316105
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Kinetic IR spectroscopy was used to reveal {beta}-sheet formation and water expulsion in the folding of single-chain monellin (SMN) composed of a five-stranded {beta}-sheet and an {alpha}-helix. The time-resolved IR spectra between 100 {micro}s and 10 s were analyzed based on two consecutive intermediates, I1 and I2, appearing within 100 {micro}s and with a time constant of {approx}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 {beta}-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 {beta}-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 C[IMG]/medium/cjs0808.gif" ALT="Formula ">O group in the {beta}-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.
