Liskin
Swint-Kruse, Ph.D.
Assistant Professor
Biochemistry and Molecular Biology
Allosteric Determinants in the Laci/Galr Family
Mentor:
Glen
Andrews
Frequently, comparable protein functions are based upon similar three-dimensional structures. These scaffolds are further "decorated" at specific locations to fine-tune the specific role of each protein. In sequence comparisons, decorative sites should be characterized by variability between homologous proteins. Unfortunately, this trait is also shared with unimportant sites that have no evolutionary pressure to be conserved. While variation is a well-recognized contributor to ligand specificity, it is also critical to other features of function, such as information transmission through the protein structure as signaling information is conveyed from one binding site to another. However, atomic-level details related to dynamic processes are very difficult to discern from static structures.
Recently, both limitations were circumvented in a multi-disciplinary study that identified four sites that appear to differentiate LacI and PurR allosteric signaling. The same four sites are predicted to be important to inter-molecular communication for most proteins in the broader LacI/GalR family. As a group, these proteins detect changing levels of small-molecule signals (effectors) and modify bacterial metabolic processes through altered transcription control. The four sites identified in LacI and PurR are located on one face of a cross-domain interface, between the effector- and DNA-binding regions.
Experiments are proposed to test the functional relevance of these positions using chimeric constructs comprising the LacI DNA-binding domain fused to the effector-binding domains of PurR, GalR, CRA and CytR. The first aim of the project is to directly test the hypothesis that chimeric proteins will not have allosteric function without further modification at the indicated sites in the LacI DNA-binding domain. The remaining research is designed to study complementary sites on the half of the interface contributed by the homologous effector-binding domains. The second aim of this research focuses on PurR, which has opposite allostery from LacI (repressible vs. inducible) and has structures available to provide specific hypotheses about residues required in allostery. Both of these first two steps can be complemented with random mutagenesis to address areas where specific hypotheses fail. Similar strategy is used in the third aim of the research and will provide information about the effector-binding domains of inducible family members with unknown structure. Results from all experiments will delineate a range of allosteric pathways supported by the common architecture and give insight to components critical to viability of pathogenic bacteria.
Dr. Swint-Kruse graduated from the COBRE in 2007 when she was awarded an NIH R-01 grant "Functional effects of exchanging domains and linkers in transcription regulators."
Copyright © 2008
Center for Biomedical Research Excellence in Protein Structure and Function
Department of Medicinal Chemistry, School of Pharmacy
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