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Refolding of urea-denatured adenylate kinase.

ABSTRACT: The refolding of urea-denatured adenylate kinase (EC 2.7.4.3) has been followed by formation of the secondary structure, change of surface hydrophobicity and recovery of catalytic activity. During refolding of adenylate kinase with a 20-80-fold dilution of 4 M urea-denatured enzyme at 10 degrees C, the formation of the secondary structure is a fast process with a rate constant of >0.16 s-1. Transient enhancement of the 8-anilino-1-naphthalenesulphonate (ANS) fluorescence intensity is followed by a fluorescence decrease to the level equal to the value characteristic of native enzyme. The desorption of ANS binding fluorescence is relatively slow and can be fitted to a first order reaction with a rate constant of 0.004 s-1 when the ANS is present in the dilution buffer. The desorption of ANS-binding fluorescence is accelerated in the presence of nucleotide substrates. The rate constants are increased to 0.049, 0. 029, 0.028 and 0.029 s-1 in the presence of 1 mM AMP, MgATP, ATP and ADP respectively. The refolding rate constant calculated from the initial fluorescence intensity after mixing ANS with protein at different refolding intervals is 0.016 s-1, which is faster than those obtained when ANS is present throughout the refolding process, indicating that the binding of ANS with a partially folded intermediate retards its further refolding to its native structure. The reactivation rate is even faster than the rates of refolding monitored in the absence of substrates, showing that the refolding is accelerated in the presence of the substrates. A possible refolding pathway and the accelerating effect of substrates are discussed.

SUBMITTER: Zhang Hj

PROVIDER: S-EPMC1219598 | biostudies-other | 1998 Jul

REPOSITORIES: biostudies-other

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Project description:Delta-crystallin is the major soluble protein in avian eye lenses with a structural role in light scattering. Dissociation and unfolding of the tetrameric protein in guanidinium chloride (GdmCl) can be sensitively monitored by the intrinsic tryptophan fluorescence. In this study refolding of GdmCl-denatured delta-crystallin was investigated. A marked hysteresis was observed while refolding by dilution of the 5 M GdmCl-denatured delta-crystallin. The secondary structure of the refolded protein was largely restored. However, monitoring intrinsic fluorescence of single tryptophan mutants indicated that the microenvironment of domain 1 (W74) was not restored. The region containing W169, which is close to the dimer interface, remained exposed following refolding. During refolding of the wild-type protein, dimeric, tetrameric, and aggregate forms were identified. The ratio of tetramer to dimer increased with time, as judged by gel-filtration chromatography and nondenaturing gel electrophoresis. However the observed levels of tetramer did not return to the same levels as observed before GdmCl treatment. The proportion of tetramer was significantly decreased in the N-25 deletion mutant and it did not increase with time. These results suggest that there is a kinetic barrier for assembly of dimers into tetramers. The consequence of this is that dimers refold to form aggregates. Aggregation seems to follow a nucleation mechanism with an apparent reaction order of 4.7+/-0.2, suggesting four or five monomers constitute the core structure of nucleus, which propagate to form high molecular weight aggregates. Addition of alpha-crystallin during refolding prevents aggregation. Thioflavin T and Congo red assays indicated a regular structure for the protein aggregates, which appear as hollow tubules packed into helical bundles. Aggregate formation was protein concentration dependent that progressed via two stages with rate constants of 0.0039+/-0.0006 and 0.00043+/-0.00003 s(-1), respectively. We propose that the N-terminal segment of delta-crystallin plays a critical role in proper double dimer assembly and also in the assembly of nucleus to aggregate formation.

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Refolding of urea-denatured adenylate kinase.

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