Structural Characterization of the M* Partly Folded Intermediate of Wild Type and P138A Aspartate Aminotransferase from Escherichia coli

Abstract

A combination of spectroscopic techniques, hydrogen/ deuterium exchange, and limited proteolysis experiments coupled to mass spectrometry analysis was used to depict the topology of the monomeric M* partly folded intermediate of aspartate aminotransferase from Escherichia coli in wild type (WT) as well as in a mutant form in which the highly conserved cis-proline at position 138 was replaced by a trans-alanine (P138A). Fluorescence analysis indicates that, although M* is an off-pathway intermediate in the folding of WT aspartate aminotransferase from E. coli, it seems to coincide with an onpathway folding intermediate for the P138A mutant. Spectroscopic data, hydrogen/deuterium exchange, and limited proteolysis experiments demonstrated the occurrence of conformational differences between the two M* intermediates, with P138A-M* being conceivably more compact than WT-M*. Limited proteolysis data suggested that these conformational differences might be related to a different relative orientation of the small and large domains of the protein induced by the presence of the cis-proline residue at position 138. These differences between the two M* species indicated that in WT-M* Pro138 is in the cis conformation at this stage of the folding process. Moreover, hydrogen/deuterium exchange results showed the occurrence of few differences in the native N 2 forms of WT and P138A, the spectroscopic features and crystallographic structures of which are almost superimposable.The mechanism by which proteins fold into their unique native structures is still a central problem in structural biology. It is increasingly recognized that the structure of non-native states of proteins can provide significant insight into fundamental issues such as the relationship between protein sequences and three-dimensional structures, the nature of protein folding pathways, the stability of proteins and their turnover in the cell, and the transport of proteins across membranes (1). Moreover, intermediate states experienced by proteins in vivo often play a major role in protein association and aggregation, leading to the "so-called" conformational diseases (2). In contrast to the large amount of structural information available on native folded proteins, however, too few partly folded intermediates have been characterized thoroughly enough to propose general models on how the native state is attained and which is the structure of transient folding states. Investigation of a wide range of non-native states would then be of considerable value. To meet this need, new analytical strategies able to characterize transient species and to define the molecular details through which diverse proteins fold are required.Recently, structural biologists have turned their attention to integrated strategies for the definition of the surface topology of proteins and protein complexes. Although these approaches provide low resolution data, they are amenable to the analysis of transient species and partly folded intermediates. Limited proteolysis and amide hydrogen ex...

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