Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue

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dc.contributor.authorZhan, HLen_AU
dc.contributor.authorTaraban, Men_AU
dc.contributor.authorTrewhella, Jen_AU
dc.contributor.authorSwint-Kruse, Len_AU
dc.date.accessioned2009-07-07T06:38:44Zen_AU
dc.date.accessioned2010-04-30T05:07:04Zen_AU
dc.date.available2009-07-07T06:38:44Zen_AU
dc.date.available2010-04-30T05:07:04Zen_AU
dc.date.issued2008-08-05en_AU
dc.date.statistics2008-08-05en_AU
dc.description.abstractMany mutations that impact protein function occur at residues that do not directly contact ligand. To understand the functional contributions from the sequence that links the DNA-binding and regulatory domains of the LacI/GalR homologues, we have created a chimeric protein (LLhP), which comprises the LacI DNA-binding domain, the LacI linker, and the PurR regulatory domain. Although DNA binding site residues are identical in LLhP and LacI, thermodynamic measurements of DNA binding affinity show that LLhP does not discriminate between alternative DNA ligands as well as LacI. In addition, small-angle scattering experiments show that LLhP is more compact than LacI. When DNA is released, LacI shows a 20 angstrom increase in length that was previously attributed to unfolding of the linker. This change is not seen in apo-LLhP, even though the linker sequences of the two proteins are identical. Together, results indicate that long-range functional and structural changes are propagated across the interface that forms between the linker and regulatory domain. These changes could be mediated via the side chains of several linker residues that contact the regulatory domains of the naturally occurring proteins, LacI and PurR. Substitution of these residues in LLhP leads to a range of functional effects. Four variants exhibit altered affinity for DNA, with no changes in selectivity or allosteric response. Another two result in proteins that bind operator DNA with very low affinity and no allosteric response, similar to LacI binding nonspecific DNA sequences. Two more substitutions simultaneously diminish affinity, enhance allostery, and profoundly alter DNA ligand selectivity. Thus, positions within the linker can be varied to modulate different aspects of repressor function. © 2008, American Chemical Societyen_AU
dc.identifier.citationZhan, H. L., Taraban, M., Trewhella, J., & Swint-Kruse, L. (2008). Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue. Biochemistry, 47(31), 8058-8069. doi:10.1021/bi800443ken_AU
dc.identifier.govdoc1452en_AU
dc.identifier.issn0006-2960en_AU
dc.identifier.issue31en_AU
dc.identifier.journaltitleBiochemistryen_AU
dc.identifier.pagination8058-8069en_AU
dc.identifier.urihttp://dx.doi.org/10.1021/bi800443ken_AU
dc.identifier.urihttp://apo.ansto.gov.au/dspace/handle/10238/1475en_AU
dc.identifier.volume47en_AU
dc.language.isoenen_AU
dc.publisherAmerican Chemical Societyen_AU
dc.subjectDNA sequencingen_AU
dc.subjectAmino acidsen_AU
dc.subjectProteinsen_AU
dc.subjectLigandsen_AU
dc.subjectSmall angle scatteringen_AU
dc.subjectGene repressorsen_AU
dc.titleSubdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologueen_AU
dc.typeJournal Articleen_AU
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