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Biomedical subjects

J Keith Joung

Publications and source records attributed to J Keith Joung.

7 recordsLinked to original sources
PubMed2006

Synthetic protein-protein interaction domains created by shuffling Cys2His2 zinc-fingers.

Cys2His2 zinc-fingers (C2H2 ZFs) mediate a wide variety of protein-DNA and protein-protein interactions. DNA-binding C2H2 ZFs can be shuffled to yield artificial proteins with different DNA binding specificities. Here we demonstrate that shuffling of C2H2 ZFs from transcription factor dimerization zinc-finger (DZF) domains can also yield two-finger DZFs with novel protein-protein interaction specificities. We show that these synthetic protein-protein interaction domains can be used to mediate activation of a single-copy reporter gene in bacterial cells and of an endogenous gene in human cells. In addition, the synthetic two-finger domains we constructed can also be linked together to create more extended, four-finger interfaces. Our results demonstrate that shuffling of C2H2 ZFs can yield artificial protein-interaction components that should be useful for applications in synthetic biology.

Binding Sites↗
PubMed2006

Counter-selectable marker for bacterial-based interaction trap systems.

Counter-selectable markers can be used in two-hybrid systems to search libraries for a protein or compound that interferes with a macromolecular interaction or to identify macromolecules from a population that cannot mediate a particular interaction. In this report, we describe the adaptation of the yeast URA3/5-FOA counter-selection system for use in bacterial interaction trap experiments. Two different URA3 reporter systems were developed that allow robust counter-selection: (i) a single copy F' episome reporter and (ii) a co-cistronic HIS3-URA3 reporter vector. The HIS3-URA3 reporter can be used for either positive or negative selections in appropriate bacterial strains. These reagents extend the utility of the bacterial two-hybrid system as an alternative to its yeast-based counterpart.

Escherichia coli↗
PubMed2005

Repression of phase-variable cup gene expression by H-NS-like proteins in Pseudomonas aeruginosa.

The cupA gene cluster of Pseudomonas aeruginosa encodes components of a putative fimbrial structure that enable this opportunistic human pathogen to form biofilms on abiotic surfaces. In P. aeruginosa, cupA gene expression is repressed by MvaT, a putative transcription regulator thought to belong to the H-NS family of nucleoid-associated proteins that typically function by repressing transcription. Here, we present evidence that MvaT controls phase-variable (ON/OFF) expression of the cupA fimbrial gene cluster. Using a directed proteomic approach, we show that MvaT associates with a related protein in P. aeruginosa called MvaU. Analysis with a bacterial two-hybrid system designed to facilitate the study of protein dimerization indicates that MvaT and MvaU can form both heteromeric and homomeric complexes, and that formation of these complexes is mediated through the N-terminal regions of MvaT and MvaU, both of which are predicted to adopt a coiled-coil conformation. We show further that, like MvaT, MvaU can repress phase-variable expression of the cupA gene cluster. Our findings suggest that fimbrial genes important for biofilm formation can be expressed in a phase-variable manner in P. aeruginosa, provide insight into the molecular mechanism of MvaT-dependent gene control, and lend further weight to the postulate that MvaT proteins are H-NS-like in nature.

Bacterial Proteins↗
PubMed2005

A combined yeast/bacteria two-hybrid system: development and evaluation.

Two-hybrid screening is a standard method used to identify and characterize protein-protein interactions and has become an integral component of many proteomic investigations. The two-hybrid system was initially developed using yeast as a host organism. However, bacterial two-hybrid systems have also become common laboratory tools and are preferred in some circumstances, although yeast and bacterial two-hybrid systems have never been directly compared. We describe here the development of a unified yeast and bacterial two-hybrid system in which a single bait expression plasmid is used in both organismal milieus. We use a series of leucine zipper fusion proteins of known affinities to compare interaction detection using both systems. Although both two-hybrid systems detected interactions within a comparable range of interaction affinities, each demonstrated unique advantages. The yeast system produced quantitative readout over a greater dynamic range than that observed with bacteria. However, the phenomenon of "autoactivation" by baits was less of a problem in the bacterial system than in the yeast. Both systems identified physiological interactors for a library screen with a cI-Ras test bait; however, non-identical interactors were obtained in yeast and bacterial screens. The ability to rapidly shift between yeast and bacterial systems provided by these new reagents should provide a marked advantage for two-hybrid investigations. In addition, the modified expression vectors we describe in this report should be useful for any application requiring facile expression of a protein of interest in both yeast and bacteria.

Amino Acid Sequence↗
PubMed2004

Allosteric inhibition of zinc-finger binding in the major groove of DNA by minor-groove binding ligands.

In recent years, two methods have been developed that may eventually allow the targeted regulation of a broad repertoire of genes. The engineered protein strategy involves selecting Cys(2)His(2) zinc finger proteins that will recognize specific sites in the major groove of DNA. The small molecule approach utilizes pairing rules for pyrrole-imidazole polyamides that target specific sites in the minor groove. To understand how these two methods might complement each other, we have begun exploring how polyamides and zinc fingers interact when they bind the same site on opposite grooves of DNA. Although structural comparisons show no obvious source of van der Waals collisions, we have found a significant "negative cooperativity" when the two classes of compounds are directed to the overlapping sites. Examining available crystal structures suggests that this may reflect differences in the precise DNA conformation, especially with regard to width and depth of the grooves, that is preferred for binding. These results may give new insights into the structural requirements for zinc finger and polyamide binding and may eventually lead to the development of even more powerful and flexible schemes for regulating gene expression.

Allosteric Regulation↗
PubMed2003

Highly specific zinc finger proteins obtained by directed domain shuffling and cell-based selection.

Engineered Cys2His2 zinc finger proteins (ZFPs) can mediate regulation of endogenous gene expression in mammalian cells. Ideally, all zinc fingers in an engineered multifinger protein should be optimized concurrently because cooperative and context-dependent contacts can affect DNA recognition. However, the simultaneous selection of key contacts in even three fingers from fully randomized libraries would require the consideration of >10(24) possible combinations. To address this challenge, we have developed a novel strategy that utilizes directed domain shuffling and rapid cell-based selections. Unlike previously described methods, our strategy is amenable to scale-up and does not sacrifice combinatorial diversity. Using this approach, we have successfully isolated multifinger proteins with improved in vitro and in vivo function. Our results demonstrate that both DNA binding affinity and specificity are important for cellular function and also provide a general approach for optimizing multidomain proteins.

Amino Acid Sequence↗