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J Inorg Biochem. 2012 Oct;115:28-35. doi: 10.1016/j.jinorgbio.2012.05.009. Epub 2012 Jun 2.

Femtomolar Zn2+ affinity of LIM domain of PDLIM1 protein uncovers crucial contribution of protein-protein interactions to protein stability.

Author information

1
Department of Protein Engineering, Faculty of Biotechnology, University of Wrocław, Tamka 2, 50-137 Wrocław, Poland.

Abstract

An individual LIM domain has approximately 55 amino acids with 8 highly conserved residues responsible for binding of two Zn(2+) into two distinct zinc finger motifs. We examined LIM domain stability of PDLIM1 protein (known also as Elfin protein), its C-terminally extended constructs as well as separate zinc fingers, and several full domain mutants in terms of Zn(2+) affinity and domain stability. Thermal denaturation, mass spectrometry, limited proteolysis, protein oxidation and circular dichroism techniques were used to determine a set of thermodynamic stability parameters. The results demonstrate unambiguously very high (femtomolar) affinity of both Zn(2+) to the conserved LIM domain (K(d)(av)=2.5×10(-14) M) and its additional elevation in the C-terminally extended domain construct (K(d)(av)=3.1×10(-15) M). We demonstrate in the example of PDLIM1 using a set of LIM protein constructs and its zinc finger peptides that stability of the entire zinc-containing domain is not only defined by the Zn(2+) coordination environment but significantly depends on the set of protein-protein interactions with the C-terminus of the protein. We discuss structural similarities of LIM domains and suggest the prolongation of the conserved LIM sequence to its C-terminal helix that has a significant impact on domain stability. We also discuss the functionality of LIM domains in terms of different physiological zinc and redox buffering capacity.

PMID:
22922308
DOI:
10.1016/j.jinorgbio.2012.05.009
[Indexed for MEDLINE]

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