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Mutational analysis of the metal sites in an LIM domain
Site-directed mutagenesis was carried out to map the residues that form the two Zn(II) sites within a LIM domain. The C-terminal LIM domain derived from the cysteine-rich protein was utilized for this analysis and is referred to as LIM2. Seven cysteinyl residues and a single histidyl residue in the...
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Published in: | The Journal of biological chemistry 1994-04, Vol.269 (15), p.11108-11113 |
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Main Authors: | , , , , , , |
Format: | Article |
Language: | English |
Subjects: | |
Citations: | Items that this one cites Items that cite this one |
Online Access: | Get full text |
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Summary: | Site-directed mutagenesis was carried out to map the residues that form the two Zn(II) sites within a LIM domain. The C-terminal
LIM domain derived from the cysteine-rich protein was utilized for this analysis and is referred to as LIM2. Seven cysteinyl
residues and a single histidyl residue in the LIM2 sequence, CX2CX17HX2CX2CX2CX17CX2C, comprise the conserved residues in
the LIM consensus that are potential Zn(II) ligands. Two Zn(II) binding sites exhibiting tetrathiolate (S4) and S3N1 Zn(II)
coordination are displayed by LIM2 (Kosa, J. L., Michelsen, J. W., Louis, H. A., Olsen, J. I., Davis, D. R., Beckerle, M.
C., and Winge, D. R. (1994) Biochemistry 33, 468-477). Site-directed mutagenesis was employed to generate three mutant LIM2
proteins with conversions of the second conserved cysteine to histidine (C2H), the fifth conserved cysteine to histidine (C5H),
and the last conserved cysteine to aspartate (C8D). Metal coordination by the mutant proteins was evaluated by atomic absorption
spectroscopy, Co(II) electronic spectroscopy, and 113Cd NMR spectroscopy. The results permit discrimination between various
models of metal ion binding and suggest that the LIM domain is comprised of a S3N1 site generated from the four N-terminal
candidate ligands (CX2CX17HX2C) and a S4 site generated from the four C-terminal candidate ligands (CX2CX17CX2C). |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1016/S0021-9258(19)78098-3 |