Robert Schleif
Research
Lab and teaching Some comments for graduate students Books Photographic Random Laboratory members Lab reunion, 2009 What important mol. bio. event took place in 1940? |
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Professor Department of Biology Johns Hopkins University 3400 N. Charles Street Baltimore, MD 21218-2685 Email: person at jhu dot edu where person is rfschleif Office 410 516-5206 Lab 410 516-5207 Departmental fax 410 516-5213 B.S.
Tufts University Ph.D. University of California, Berkeley Postdoctoral Harvard University |
RESEARCH INTERESTSResearch in my laboratory is 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 we have a large collection of mutations in AraC and the regulatory region as well as many mutant DNA's and proteins, many of our ongoing studies use this system. 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. More recently we discovered the two domain structure to AraC and grew the crystals from which the structure of the dimerization domain was determined. This work in connection with biochemical and genetic studies led us to the discovery of the role of the N-terminal arms on AraC and the "light switch" mechanism by which the arms of the protein regulate the looping-unlooping activity of the protein. The light switch mechanism and the use of arms in domain-domain and protein-protein interactions may be widespread in nature, and we are examinining its occurrence. Recently we demonstrated that the light switch mechanism can be ported to other proteins, and we have constructed a b-galactosidase whose activity is controlled by the light switch mechanism from AraC. The enzyme's activity is modulated by the presence of arabinose. Additionally, we have constructed other and simpler "man-made" regulatory proteins. Several years ago we found that the DNA binding domain
of AraC
may
readily be
overproduced and purified. It appears to be a very good material
for NMR studies, and we are now determining its structure by NMR as
well
as mapping its interactions with other proteins and with DNA.
Current work is also directed towards improving our understanding of
the electrostatics of protein-DNA interactions, what controls which
of alternative structures the chameleon-like N-terminal arm of
AraC assumes, the basic forms of allsteric regulation displayed by gene
regulatory proteins, and the physical basis of altered properties of
AraC mutants. Approaches 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 is AraC protein. Frequently we develop 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 are now developing a method for investigation of the very weak protein-protein and domain-domain interactions that are often found in complex regulatory systems.SUMMARY OF THE REGULATION MECHANISM OF THE ARABINOSE OPERON
 AraC protein consists of two loosely connected domains, a DNA-binding domain that both binds to the various I-like sites and which also interacts with RNA polymerase to activate transcription, and a dimerization domain that also binds arabinose. AraC protein is caused to form the DNA loop between the I1 and O2 half-sites by the N-terminal arms that extend from the dimerization domains and bind to the back of the DNA binding domains. The simultaneous interaction of these arms with both the dimerization domains and the DNA binding domains holds the DNA binding domains in a relative orientation that energetically favors DNA loop formation and disfavors binding to the direct repeat I1 and I2 half-sites. Upon the binding of arabinose to the dimerization domains, however, the N-terminal arms restructure such that the DNA binding domains are released and are thus freed to assume any relative orientation they like. As a result, they now prefer to bind to the two adjacent, half-sites I1 and I2, where such binding activates transcription from pBAD. RECENT PUBLICATIONSUpdated 9/09
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