Triticum durum metallothionein. Isolation of the gene and structural characterization of the protein using solution scattering and molecular modeling.
A novel gene sequence, with two exons and one intron, encoding a metallothionein (MT) has been identified in durum wheat Triticum durum cv. Balcali85 genomic DNA. Multiple alignment analyses on the cDNA and the translated protein sequences showed that T. durum MT (dMT) can be classified as a type 1 MT. dMT has three Cys-X-Cys motifs in each of the N- and C-terminal domains and a 42-residue-long hinge region devoid of cysteines. dMT was overexpressed in Escherichia coli as a fusion protein (GSTdMT), and bacteria expressing the fusion protein showed increased tolerance to cadmium in the growth medium compared with controls. Purified GSTdMT was characterized by SDS- and native-PAGE, size exclusion chromatography, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. It was shown that the recombinant protein binds 4 +/- 1 mol of cadmium/mol of protein and has a high tendency to form stable oligomeric structures. The structure of GSTdMT and dMT was investigated by synchrotron x-ray solution scattering and computational methods. X-ray scattering measurements indicated a strong tendency for GSTdMT to form dimers and trimers in solution and yielded structural models that were compatible with a stable dimeric form in which dMT had an extended conformation. Results of homology modeling and ab initio solution scattering approaches produced an elongated dMT structure with a long central hinge region. The predicted model and those obtained from x-ray scattering are in agreement and suggest that dMT may be involved in functions other than metal detoxification.
Bilecen
Kivanc
K
Sabanci University, Faculty of Engineering and Natural Sciences, 34956, Orhanli, Tuzla, Istanbul, Turkey.
Ozturk
Umit H
UH
Duru
Adil D
AD
Sutlu
Tolga
T
Petoukhov
Maxim V
MV
Svergun
Dimitri I
DI
Koch
Michel H J
MH
Sezerman
Ugur O
UO
Cakmak
Ismail
I
Sayers
Zehra
Z
D000595:Amino Acid Sequence; D001483:Base Sequence; D002104:Cadmium / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid; D014908:Triticum / Q000737:chemistry* / Q000235:genetics*
D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D002104:Cadmium; D008668:Metallothionein