Characterization and sequential localization of the metal clusters in sea urchin metallothionein.
The mode of metal binding in sea urchin metallothionein (MT) was explored by electronic absorption, chiroptical, NMR, and mass spectroscopic methods. Recombinant sea urchin MT containing 7 equiv of the natural mixture of Cd isotopes was stripped of the metal by exposure to low pH and reconstituted with 113Cd (> 95% enriched). Comparison of the electronic spectroscopic and chiroptical features and the 113Cd NMR spectra of the reconstituted material with those of the native recombinant material indicated that the reconstituted material had regained the native conformation. The shoulder at 250 nm in the electronic absorption spectrum, the biphasic circular dichroism profile centered at 250 nm, and the chemical shift positions (605-695 ppm) of the seven 113Cd NMR resonances all strongly suggested that sea urchin MT like all other well characterized MTs contains clusters made up of tetrahedral Cd-thiolate units. The 113Cd chemical shift correlation spectrum of the reconstituted protein proved the existence of such metal clusters and allowed the unambiguous assignment of some of the metal connectivities. Homonuclear decoupling experiments in which Cd resonances were selectively saturated indicated moreover a partitioning of the metal complement into two separate clusters containing three and four Cd ions. The same proposition was supported by the selective reduction of three 113Cd resonances upon partial metal depletion following exposure of the protein to EDTA. Thus, the three-metal cluster is energetically less stable than the four-metal cluster.(ABSTRACT TRUNCATED AT 250 WORDS)
Wang
Y
Y
Biochemisches Institut, Universität Zürich, Switzerland.
Mackay
E A
EA
Zerbe
O
O
Hess
D
D
Hunziker
P E
PE
Vasák
M
M
Kägi
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D002104:Cadmium; D004492:Edetic Acid; D007554:Isotopes; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D008668:Metallothionein / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012617:Sea Urchins; D013053:Spectrophotometry; D013054:Spectrophotometry, Atomic; D013381:Subtilisins; D013438:Sulfhydryl Compounds
D007554:Isotopes; D010446:Peptide Fragments; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D002104:Cadmium; D008668:Metallothionein; D004492:Edetic Acid; D013381:Subtilisins