Structural characterization of the M* partly folded intermediate of wild type and P138A aspartate aminotransferase from Escherichia coli.
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.
Birolo
Leila
L
Dipartimento di Chimica Organica e Biochimica, Università Federico II di Napoli, Complesso Universitario Monte Sant'Angelo, Via Cinthia, 80126 Napoli, Italy. birolo@unina.it
Dal Piaz
Fabrizio
F
Pucci
Piero
P
Marino
Gennaro
G
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
D011392:Proline; D001219:Aspartate Aminotransferases; D002918:Chymotrypsin