Conformational changes and in vitro core-formation modifications induced by site-directed mutagenesis of the specific N-terminus of pea seed ferritin.
Plant ferritin has a three-dimensional structure predicted to be very similar to that of animal ferritin. It has, however, an additional specific sequence of 24 amino acids at its N-terminus named extension peptide (EP). In order to determine precisely the interactions between EP and other domains of the pea seed ferritin subunit, three point mutations were performed. The mutated residues were chosen by three-dimensional computer modelling of the pea seed ferritin subunit structure [Lobréaux, Yewdall, Briat and Harrison (1992) Biochem. J. 228, 931-939]. The mutant recombinant proteins were expressed in Escherichia coli and purified to homogeneity; all the mutants were found to be assembled as 24-mers. When Ala-13 was replaced by His, as in mammalian ferritins, ferroxidase activity was significantly reduced. Moreover, in vitro iron-core formation in Pro-X-->Ala, Lys-R-->Glu and Ala-13-->His mutants was increased after denaturation by urea followed by renaturation; this was also observed with the EP deletion mutant (r delta TP/EP). The recombinant ferritins were also analysed using tryptophan fluorescence spectra. The r delta TP/EP, Pro-X-->Ala and Lys-R-->Glu mutants were found to be more susceptible to denaturation by urea than the native r delta TP pea seed ferritin.
Laboratoire de Biologie Moléculaire Végétale, Centre National de la Recherche Scientifique (Unité de Recherche 1178), Grenoble, France.
O
O
van Wuytswinkel
J F
JF
Briat
D000595:Amino Acid Sequence; D001483:Base Sequence; D005293:Ferritins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D018532:Pisum sativum; D010446:Peptide Fragments / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012639:Seeds / Q000737:chemistry*; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D014508:Urea
D010446:Peptide Fragments; D010940:Plant Proteins; D011994:Recombinant Proteins; D014508:Urea; D005293:Ferritins