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Quenched-flow hydrogen exchange labeling, monitored by 1H NMR and electrospray ionization mass spectrometry (ESI-MS), has been employed in conjunction with stopped-flow circular dichroism and fluorescence to study the kinetic refolding from guanidinium chloride of a derivative of hen lysozyme in which one of the four disulfide linkages (Cys6-Cys127) has been selectively chemically reduced and carboxymethylated (CM6,127-lysozyme). Removal of this disulfide bridge has little effect on the structure and activity of the native enzyme, and the overall kinetics of refolding are very similar to those of the unmodified protein. A substantial amount of secondary structure is formed within 2 ms of the initiation of folding, followed by the slower formation of tertiary interactions characteristic of the native state, which are attained with a time constant (tau) of ca. 200 ms. There is clear evidence for fast and slow refolding populations, as in the intact protein. Folding of the three-disulfide derivative does, however, exhibit a major difference from that of the intact protein under the same final refolding conditions, in that the transient intermediate on the major refolding pathway of the intact protein, having persistent structure in the alpha-helical domain of the protein, is not detected by hydrogen exchange labeling during folding of the three-disulfide derivative. This suggests that the disulfide bond linking the N- and C-terminal regions of the protein is crucial for stabilization of the partially folded intermediate. In addition, the overshoot in the far-UV CD and the fluorescence minimum, both of which are attributed to non-native interactions, is not observed in the folding of CM6,127-lysozyme. That the lack of a detectable stable intermediate in the folding of CM6,127-lysozyme does not significantly affect the rate of attainment of the native state of the protein supports the proposed independent nature of the two folding domains and, as the Cys6-Cys127 disulfide bond is located in the alpha-domain, indicates that the rate-limiting step in folding of the intact protein, as well as of the three-disulfide derivative, involves stabilization of the beta-domain. The role of disulfide bridges in the formation and maintenance of the three-dimensional fold of proteins and in facilitating the observation of marginally stable intermediate species is discussed.


Journal article



Publication Date





13038 - 13048


Animals, Biological Transport, Chickens, Circular Dichroism, Cysteine, Disulfides, Female, Hydrogen, Kinetics, Magnetic Resonance Spectroscopy, Mass Spectrometry, Muramidase, Protein Conformation, Protein Folding, Reference Standards, Spectrometry, Fluorescence