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Research in my laboratory has been directed at understanding the basic biochemical and biophysical principles involved in protein function through the combined use of biochemistry, genetics, genetic engineering, and biophysics. Our criterion for understanding is that we can design and build systems that actually work and make use of these principles. Since we have had extensive experience with the arabinose operon and systems related to it and developed a large collection of mutations in AraC and the regulatory region as well as many mutant DNA's and proteins. The ara system permits economic and rapid handling of the biology while displaying most of the repertoire of protein-protein, protein-DNA and gene regulatory principles that are found in prokaryotes and eukaryotes.
In 1984 we made the original discovery of DNA looping, a mechanism now known to be widely used in biology. Later we discovered the two domain structure to AraC and grew the crystals from which the structure of the dimerization domain in the presence and absence of arabinose was determined. This work in connection with biochemical and genetic studies led to the discovery of the role of the N-terminal arms on AraC and the mechanism where the two positions of the arms of the protein regulate the looping-unlooping activity of the protein. We demonstrated that a version of the mechanism can be ported to other proteins, and we have constructed a β-galactosidase whose activity is controlled by the mechanism from AraC. The enzyme's activity is modulated by the presence of arabinose.
We have found that the DNA binding domain of AraC may readily be overproduced and purified. It was a very good material for NMR studies, and we determined its structure by NMR. One recent objective was to determine precisely which residues are in contact between the dimerization and DNA binding domains both in the absence of arabinose and in the presence of arabinose. Genetic, and biochemical, methods were used to determine this. Recently we found that it is the helicity of the interdomain linker that is controlled by the presence or absence of arabinose bound to AraC. In the absence of arabinose, the linker is helical, and in the presence of arabinose, it is a random coil.
Approaches that were commonly used in the laboratory include biochemistry, genetics, genetic engineering, physiological measurement, and biochemical and physical-chemical approaches, for example crystallography, fluorescence, electrophoresis, plasmon resonance, NMR, as well as computational approaches. Our primary, but not only, subject for comparison of theory and experiment was AraC protein.
Frequently we developed new experimental techniques to facilitate our studies. In the past we developed the DNA migration retardation assay so that biochemically meaningful information could be obtained from it and developed the missing contact method for determining specific amino acid-base interactions in DNA. More recently we developed methods for: locating linker regions in multi-domain proteins, constructing functional chimeric proteins when the domain locations are unknown, precise comparison of DNA binding affinities, and refolding DNA-binding proteins from insoluble inclusion bodies. We also developed a method for investigation of the very weak protein-protein and domain-domain interactions that are often found in complex regulatory systems.
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Frontiers in molecular biosciences (2022): 848444-848444
Biochemistryno. 26 (2019): 2875-2882
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Biochemistryno. 26 (2019): 2867-2874
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Proteins: Structure, Function, and Bioinformaticsno. 4 (2016): 448-460
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mag(2014)
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