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Structural and kinetic characterization of early folding events in β-lactoglobulin

Nature Structural Biology volume 8, pages 151–155 (2001)Cite this article

Abstract

We have defined the structural and dynamic properties of an early folding intermediate of β-lactoglobulin known to contain non-native α-helical structure. The folding of β-lactoglobulin was monitored over the 100 μs–10 s time range using ultrarapid mixing techniques in conjunction with fluorescence detection and hydrogen exchange labeling probed by heteronuclear NMR. An initial increase in Trp fluorescence with a time constant of 140 μs is attributed to formation of a partially helical compact state. Within 2 ms of refolding, well protected amide protons indicative of stable hydrogen bonded structure were found only in a domain comprising β-strands F, G and H, and the main α-helix, which was thus identified as the folding core of β-lactoglobulin. At the same time, weak protection (up to ∼10-fold) of amide protons in a segment spanning residues 12–21 is consistent with formation of marginally stable non-native α-helices near the N-terminus. Our results indicate that efficient folding, despite some local non-native structural preferences, is insured by the rapid formation of a native-like α/β core domain.

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Figure 1: Folding kinetics of βLG monitored by fluorescence.
Figure 2: Proton occupancy measured by quenched D-H exchange for representative amide protons in various secondary structural elements of βLG plotted versus refolding time (logarithmic scale).
Figure 3: Amide protection results.
Figure 4: Schematic diagram of a subset of the secondary structure of βLG, including the core region of the β-sheet (βF, βG and βH), βA, and the major α-helix (shown as a helical wheel).
Figure 5: Schematic representation of the conformational states encountered during folding of βLG, including the unfolded ensemble, a partially structured intermediate populated on the millisecond time scale and the native state (ribbon diagram based on the X-ray structure24; PDB code 3BLG).

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Acknowledgements

We thank S. Era for assistance and M. Taguchi for protein preparation, and J.M. Sauder and R.L. Dunbrack for critical reading of the manuscript. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan (to K.K. and Y.G.), grants from the National Institutes of Health and the National Science Foundation (to H.R.), and an appropriation from the Commonwealth of Pennsylvania to the Institute for Cancer Research. The NMR facility of Fox Chase Cancer Center was supported by a grant from the Kresge foundation.

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Author notes

  1. Kazuo Kuwata, Ramachandra Shastry and Heinrich Roder: These authors contributed equally.

Authors and Affiliations

  1. Department of Physiology, School of Medicine, Gifu University, Tsukasa-machi, 500-8705, Gifu, Japan

    Kazuo Kuwata

  2. Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, 19111, Pennsylvania, USA

    Ramachandra Shastry, Hong Cheng & Heinrich Roder

  3. Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, 565-0871, Osaka, Japan

    Masaru Hoshino & Yuji Goto

  4. Department of Food Science and Nanobiotechnology Center, Cornell University, Ithaca, 14853, New York, USA

    Carl A. Batt

  5. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Heinrich Roder

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  1. Kazuo Kuwata
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Correspondence to Yuji Goto or Heinrich Roder.

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Kuwata, K., Shastry, R., Cheng, H. et al. Structural and kinetic characterization of early folding events in β-lactoglobulin. Nat Struct Mol Biol 8, 151–155 (2001). https://doi.org/10.1038/84145

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