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Stepwise formation of α-helices during cytochrome c folding

Nature Structural Biology volume 7, pages 514–520 (2000)Cite this article

Abstract

Two models have been proposed to describe the folding pathways of proteins. The framework model assumes the initial formation of the secondary structures whereas the hydrophobic collapse model supposes their formation after the collapse of backbone structures. To differentiate between these models for real proteins, we have developed a novel CD spectrometer that enables us to observe the submillisecond time frame of protein folding and have characterized the timing of secondary structure formation in the folding process of cytochrome c (cyt c). We found that ∼20% of the native helical content was organized in the first phase of folding, which is completed within milliseconds. Furthermore, we suggest the presence of a second intermediate, which has α-helical content resembling that of the molten globule state. Our results indicate that many of the α-helices are organized after collapse in the folding mechanism of cyt c.

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Figure 1: Schematic drawing of the rapid solution mixer developed for the measurements of transient CD spectra.
Figure 2: The mixing profile of the developed rapid solution mixer.
Figure 3: Comparison of the CD spectra obtained using the developed mixer and a standard cell.
Figure 4: The time resolved CD spectra of the refolding process of cyt c initiated by a rapid jump in pH from 2.0 to 4.5.
Figure 5: CD spectra of intermediate I observed under different experimental conditions.
Figure 6: The time courses of the mean residue ellipticity observed at 222 nm (θ222) for the refolding experiments of cyt c conducted under different conditions.
Figure 7

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Acknowledgements

Supported by Grants-in-Aids for Scientific Research from the Ministry of Education, Science, Sports and Culture to S.T., K.I. and I.M.

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Authors and Affiliations

  1. Department of Molecular Engineering, Graduate School of Engineering, Kyoto University, Sakyo, 606-8501, Kyoto, Japan

    Shuji Akiyama, Satoshi Takahashi, Koichiro Ishimori & Isao Morishima

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  1. Shuji Akiyama
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Correspondence to Isao Morishima.

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Akiyama, S., Takahashi, S., Ishimori, K. et al. Stepwise formation of α-helices during cytochrome c folding. Nat Struct Mol Biol 7, 514–520 (2000). https://doi.org/10.1038/75932

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