Department of Biochemistry and Molecular Biology Areas of research emphasis: Protein structure-function; protein engineering
One outcome of the genomic revolution is that thousands of protein sequences have been grouped into families based upon their sequence similarities. Within families, homologous proteins usually have similar structures and perform similar functions. Surprisingly, multiple homologues with the same general structure/function can be present within a single cell. Nonetheless, each homologue performs a specific, distinct function that is critical to maintaining overall health. Therefore, some of the sequence differences between family members must be important for conveying unique function. Identification of these “specificity determining” sites is important to predicting propensity for some genetic diseases and for engineering novel protein functions desirable in biotechnology.
We are developing strategies to identify specificity determinants that are hidden amongst a background of unimportant residues. Our current model system is the LacI/GalR family, whose proteins control transcriptional response for many bacterial processes. All bacteria have LacI/GalR homologues; E. coli have 16. Several pathogens lose virulence if family members CRA and CcpA are disabled. The LacI/GalR proteins have the common function of transcription regulation via DNA-binding. The latter is modulated by protein binding to regulatory molecules that are available only under certain environmental conditions. To carry out their unique functions, individual homologues recognize distinct DNA and regulatory ligands. In the Swint-Kruse lab, two projects are in progress to identify specificity determinants for this family – one experimental and one computational.
EXPERIMENTAL: One of the common approaches to protein engineering is to recombine domains and thus create novel combinations of functions. However, if the domain-domain interface contains specificity determinants, the new protein could have unanticipated functional properties. We are identifying the specificity determinants that are present in the interface between the DNA-binding and regulatory domains of the LacI/GalR proteins. Our general strategy is to exchange domains between family members and use tools from biology and biochemistry to identify which positions convey various unique functions to individual proteins. Results will be used to develop rules for reliably recombining domains in this family and to evaluate and improve bioinformatics analyses of protein families in general.
COMPUTATIONAL: The regulatory domains of LacI/GalR transcription control proteins are also structurally homologous to the periplasmic binding proteins and extracellular domains of some G-protein coupled receptors. All of these families are involved in signaling processes and utilize the same general strategy - bind a small molecule in a central cleft and propagate the message to another part of the protein. Differences between the proteins include: (1) The small molecule signal is different for each protein. (2) The signaling pathways through the protein structure as well as the final molecular location of the information differs both within and between the protein families. Targeted molecular dynamics (TMD) is being used to simulate motions in the proteins as the message is propagated from the binding site to other regions of the protein. Data from homologues will be compared and contrasted to identify the range of signaling pathways supported by the common underlying protein fold. Ultimately, we will use this information to construct homologues with novel signaling functions.
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Databases of LacI Variants
Structural Effects of LacI Variants
Functional Effects of LacI Variants
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