Related Experiment Video
Updated: Aug 17, 2026

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains
Published on: March 25, 2015
Zinc and DNA binding properties of a novel LIM homeodomain protein Isl-2
Abstract:
LIM homeodomain proteins are a family of recently characterized proteins which contain, in addition to a homeodomain, two tandem repeats of conserved Cys-His motifs termed as LIM domains. We have recently isolated several clones from a chinook salmon pituitary cDNA library that encode two novel LIM homeodomain proteins, Isl-2 and Isl-3, which are structurally related to rat Isl-1. In the present study, we used the salmon Isl-2 to determine the role of LIM domains in DNA binding. Several glutathione S-transferase (GST) fusion proteins containing either full length Isl-2 or various portions of this protein were expressed in bacteria. Zinc blot analysis reveals that the LIM domains produced in bacteria are capable of binding zinc. Gel shift analysis indicates that all homeodomain-containing fusion proteins are able to bind to a TAAT target sequence while the fusion proteins containing only the LIM domain are not. In contrast to a previous observation that the LIM domains of rat Isl-1 have an inhibitory role in DNA binding, full length salmon Isl-2 containing both the LIM domains and a homeodomain can bind to a TAAT target sequence. To further examine the role of LIM domains in DNA binding, several GST fusion proteins were used to select specific target DNA sequences from a pool of randomly incorporated oligonucleotides. Specific target DNAs were selected by fusion proteins containing the homeodomain or the full length Isl-2, but not by LIM domain only fusion proteins, indicating that the LIM domain alone is not involved in DNA binding. The selected target DNAs were cloned and sequenced. They revealed two classes of consensus, C/TTAATG/TG/A and C/TTAAGTG, for both the homeodomain and full length Isl-2. The two classes of consensus competed with each other for binding to the homeodomain. The equilibrium dissociation constants for DNA binding, estimated by Scatchard analysis, were similar for the homeodomain and full length Isl-2.(ABSTRACT TRUNCATED AT 400 WORDS)
Related Concept Videos
Conservation of Protein Domains Over Different Proteins
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to form...
Conserved Binding Sites
Binding sites are often located in large pockets, and if their location on a protein’s surface is unknown, it can be predicted using various approaches. The energetic method computationally analyses the...
Cooperative Binding of Transcription Regulators
Co-activators and Co-repressors
Eukaryotic Transcription Inhibitors
Eukaryotic transcription inhibitors usually contain two distinct domains, a DNA...
Single-Strand DNA Binding Proteins

