The Cu(I)(7) cluster in yeast copper thionein survives major shortening of the polypeptide backbone as deduced from electronic absorption, circular dichroism, luminescence and( 1)H NMR.
Owing to the frustrating experience of not being able to obtain crystalline yeast Cu(I)(7) -metallothionein, thereby allowing elucidation of the X-ray structure, truncated forms were prepared to facilitate possible crystallization. The mobile remnants at either the N- or C-terminal end of the polypeptide chain were omitted. In parallel with the crystallization efforts, it was of interest to examine the degree to which the shortening of the protein portion might affect the intactness of the Cu(I)(7) -thiolate cluster, thereby hampering their use as structural models for the intact protein. (1)H two-dimensional NMR spectroscopy at 800 MHz was performed on the intact wild-type yeast Cu(7)-thionein and on two truncated forms (peptide(-1-40) and peptide(5-40)). The NMR spectral data reveal, regardless of the length of the polypeptide chain, that the spin patterns were fully preserved with all relevant NOEs. The corresponding calculated structures were virtually identical. All other spectrometric properties, including circular dichroism, luminescence and electronic absorption, allowed the same conclusion. Minor differences were observed in the chiroptic and luminescent measurements. Interestingly, however, the resistance towards oxygen was progressively diminished with decreasing length of the polypeptide backbone. The half-life of the luminescence of the wild-type protein was 48 h while the luminescence of the shortest peptide levelled off within 24 h.
Luchinat
Claudio
C
Anorganische Biochemie, Physiologisch-chemisches Institut, Universität Tübingen, Germany.
Dolderer
Benedikt
B
Del Bianco
Cristina
C
Echner
Hartmut
H
Hartmann
Hans-Jürgen
HJ
Voelter
Wolfgang
W
Weser
Ulrich
U
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D008163:Luminescent Measurements; D008668:Metallothionein / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010100:Oxygen / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D013053:Spectrophotometry / Q000379:methods; D015003:Yeasts
D010446:Peptide Fragments; C007121:copper thionein; D003300:Copper; D008668:Metallothionein; D010100:Oxygen