Abstract
The refolding of mitochondrial aspartate aminotransferase (mAAT; EC 2.6.1.1) has been studied following unfolding in 6 m guanidine hydrochloride for different periods of time. Whereas reactivation of equilibrium-unfolded mAAT is sigmoidal, reactivation of the short term unfolded protein displays a double exponential behavior consistent with the presence of fast and slow refolding species. The amplitude of the fast phase decreases with increasing unfolding times (k approximately 0.75 min(-1) at 20 degrees C) and becomes undetectable at equilibrium unfolding. According to hydrogen exchange and stopped-flow intrinsic fluorescence data, unfolding of mAAT appears to be complete in less than 10 s, but hydrolysis of the Schiff base linking the coenzyme pyridoxal 5'-phosphate (PLP) to the polypeptide is much slower (k approximately 0.08 min(-1)). This implies the existence in short term unfolded samples of unfolded species with PLP still attached. However, since the disappearance of the fast refolding phase is about 10-fold faster than the release of PLP, the fast refolding phase does not correspond to folding of the coenzyme-containing molecules. The fast refolding phase disappears more rapidly in the pyridoxamine and apoenzyme forms of mAAT, both of which lack covalently attached cofactor. Thus, bound PLP increases the kinetic stability of the fast refolding unfolding intermediates. Conversion between fast and slow folding forms also takes place in an early folding intermediate. The presence of cyclophilin has no effect on the reactivation of either equilibrium or short term unfolded mAAT. These results suggest that proline isomerization may not be the only factor determining the slow refolding of this cofactor-dependent protein.
Highlights
The folding and unfolding of proteins are complex processes affected by a number of features such as size, chain topology, and oligomeric state and by the amino acid sequence, which encodes each unique three-dimensional structure [1]
Reactivation Kinetics of pyridoxal 5-phosphate (PLP)-mitochondrial aspartate aminotransferase (mAAT) after Different Times of Unfolding—It has been reported that the rate of refolding of chicken cAAT and mAAT decreased by a factor of 4 and 25, respectively, when the proteins were denatured in 6 M guanidine hydrochloride (GdnHCl) for 40 s instead of for 40 min [29]
These results were interpreted as indicating that proline isomerization is the ratelimiting step in the slow refolding of these enzymes unfolded for long periods of time
Summary
The folding and unfolding of proteins are complex processes affected by a number of features such as size, chain topology, and oligomeric state and by the amino acid sequence, which encodes each unique three-dimensional structure [1]. We cannot discard the involvement of proline isomerization on the slow refolding steps of this protein, our results are consistent with additional structural rearrangements occurring both in the unfolded state and in populations of folding intermediates along the folding pathway.
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