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Changes in side chain packing during apomyoglobin folding characterized by pulsed thiol-disulfide exchange

Nature Structural Biology volume 5, pages 730–737 (1998)Cite this article

Abstract

It is clear that close-packed side chain interactions play a dominant role in stabilizing native proteins, but the extent to which they stabilize kinetic intermediates and shape the energetic landscape of folding is not known. A method for characterizing structural changes at the level of individual side chains is presented and applied to study the refolding of apomyoglobin mutants containing engineered cysteine residues at key helical packing interfaces. The formation of buried side chain structure at the probe sites is followed by the extent of thiol-disulfide exchange during a pulse of thiol labeling reagent (either methyl methanethiosulfonate or 5,5'-dithiobis (2-nitrobenzoic acid)) applied at various stages of folding. The results suggest that the eight helices pack in at least three distinct stages, involving formation of two intermediates with time constants of <2 ms and 50 ms. In some parts of the refolding protein, stable side chain structure can be attained very rapidly, possibly in advance of backbone hydrogen bond formation as detected by previous pulsed amide hydrogen exchange experiments.

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Figure 1: Locations of introduced cysteine residues in Mb. Helices are lettered A–H.
Figure 2: pH-induced equilibrium unfolding of apoMb monitored by Trp fluorescence.
Figure 3: a, b, Urea-induced equilibrium unfolding of apoMb monitored by Trp fluorescence.
Figure 4: Kinetics of thiol-disulfide exchange protection at 4 °C.
Figure 5: Simulation of thiol-disulfide exchange protection for a, S108C and b, A110C using equation 4.

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Acknowledgements

We thank R. Baldwin for providing a preprint of ref. 20 in advance of publication, C. Rohl, A. Martonosi and R. Cross for insightful discussions, and A. Martonosi for use of his spectrofluorometer. This work was supported by a grant from the Hendrick's Fund for Medical Research.

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  1. Department of Biochemistry and Molecular Biology, SUNY Health Science Center at Syracuse, 750 E. Adams Street, Syracuse, 13210, New York, USA

    Jeung-Hoi Ha & Stewart N. Loh

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Correspondence to Stewart N. Loh.

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Ha, JH., Loh, S. Changes in side chain packing during apomyoglobin folding characterized by pulsed thiol-disulfide exchange. Nat Struct Mol Biol 5, 730–737 (1998). https://doi.org/10.1038/1436

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