title: 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 on-pathway 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.
authors.affiliation: Dipartimento di Chimica Organica e Biochimica, Università Federico II di Napoli, Complesso Universitario Monte Sant'Angelo, Via Cinthia, 80126 Napoli, Italy. birolo@unina.it
authors.forename: Leila
authors.initials: L
authors.lastname: Birolo
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Dal Piaz
authors.forename: Piero
authors.initials: P
authors.lastname: Pucci
authors.forename: Gennaro
authors.initials: G
authors.lastname: Marino
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001219:Aspartate Aminotransferases / Q000235:genetics / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011392:Proline / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence
chemical_list: D011392:Proline; D001219:Aspartate Aminotransferases; D002918:Chymotrypsin