ANSTO Publications Online >
Journal Publications >
Journal Articles >

Please use this identifier to cite or link to this item: http://apo.ansto.gov.au/dspace/handle/10238/1475

Title: Subdividing repressor function: DNA binding affinity, selectivity, and allostery can be altered by amino acid substitution of nonconserved residues in a LacI/GalR homologue.
Authors: Zhan, HL
Taraban, M
Trewhella, J
Swint-Kruse, L
Keywords: DNA Sequencing
Amino Acids
Proteins
Ligands
Small Angle Scattering
Gene Repressors
Issue Date: 5-Aug-2008
Publisher: American Chemical Society
Citation: Zhan, 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.
Abstract: Many 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 Society
URI: http://dx.doi.org/10.1021/bi800443k
http://apo.ansto.gov.au/dspace/handle/10238/1475
ISSN: 0006-2960
Appears in Collections:Journal Articles

Files in This Item:

There are no files associated with this item.

Items in APO are protected by copyright, with all rights reserved, unless otherwise indicated.

 

Valid XHTML 1.0! DSpace Software Copyright © 2002-2010  Duraspace - Feedback