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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References
Kim, P.S. & Baldwin, R.L. Annu. Rev. Biochem. 59, 631–660 (1990).
Baldwin, R.L. & Rose, G.D. Trends Biochem. Sci. 24, 26–33 (1999).
Baldwin, R.L. & Rose, G.D. Trends Biochem. Sci. 24, 77–83 (1999).
Bryngelson, J.D., Onuchic, J.N., Socci, N.D. & Wolynes, P.G. Proteins 21, 167–195 (1995).
Dill, K.A. et al. Protein Sci. 4, 561–602 (1995).
Matthews, C.R. Annu. Rev. Biochem. 62, 653–683 (1993).
Roder, H. & Colón, W. Curr. Opin. Struct. Biol. 7, 15–28 (1997).
Kuwajima, K., Yamaya, H., Miwa, S., Sugai, S. & Nagamura, T. FEBS Lett. 221, 115–118 (1987).
Hamada, D., Segawa, S.-I. & Goto, Y. Nature Struct. Biol. 3, 868–873 (1996).
Kuwajima, K., Yamaya, H. & Sugai, S. J. Mol. Biol. 264, 806–822 (1996).
Nishikawa, K. & Noguchi, T. Methods Enzymol. 202, 31–44 (1991).
Kuroda, Y., Hamada, D., Tanaka, T. & Goto, Y. Folding Des. 1, 255–263 (1996).
Cohen, F.E. J. Mol. Biol. 293, 313–320 (1999).
Mihara, H. & Takahashi, Y. Curr. Opin. Struct. Biol. 7, 501–508 (1997).
Forge, V. et al. J. Mol. Biol. 296, 1039–1051 (2000).
Shastry, M.C.R., Luck, S.D. & Roder, H. Biophys. J. 74, 2714–2721 (1998).
Shastry, M.C.R. & Roder, H. Nature Struct. Biol. 5, 385–392 (1998).
Roder, H., Elöve, G.A. & Englander, S.W. Nature 335, 700–704 (1988).
Udgaonkar, J.B. & Baldwin, R.L. Nature 335, 694–699 (1988).
Gladwin, S.T. & Evans, P.A. Folding Des. 1, 407–417 (1996).
Sauder, J.M. & Roder, H. Folding Des. 3, 293–301 (1998).
Roder, H., Elöve, G.A. & Shastry, R.M.C. In Mechanisms of protein folding (ed. Pain, R.H.) 65–104 (Oxford University Press, New York; 2000).
Brownlow, S. et al. Structure 5, 481–495 (1997).
Qin, B.Y. et al. Biochemistry 37, 14014–14023 (1998).
Cho, Y., Batt, C.A. & Sawyer, L. J. Biol. Chem. 269, 11102–11107 (1994).
Roder, H. & Wüthrich, K. Proteins 1, 34–42 (1986).
Bai, Y., Milne, J.S. & Englander, S.W. Proteins 17, 75–86 (1993).
Woodward, C. Trends Biochem. Sci. 18, 359–360 (1993).
Bai, Y., Sosnick, T.R., Mayne, L. & Englander, S.W. Science 269, 192–197 (1995).
Raschke, T.M. & Marqusee, S. Nature Struct. Biol. 4, 298–304 (1997).
Arai, M. et al. J. Mol. Biol. 275, 149–162 (1998).
Kuwata, K. et al. Protein Sci 8, 2541–2545 (1999).
Kim, T.R. et al. Protein Eng. 10, 1339–1345 (1997).
Bax, A., Griffey, R.H. & Hawkins, B.L. J. Magn. Reson. 55, 301 (1983).
Mori, S., Abeygunawardana, C., Johnson, M.O. & van Zijl, P.C. J. Magn. Reson. B 108, 94–98 (1995).
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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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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DOI: https://doi.org/10.1038/84145
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