Robert Schleif Professor Department of Biology  Graduate Program Faculty B.S. Tufts University  Ph.D. University of California, Berkeley  Postdoctoral Harvard University

Department of Biology Johns Hopkins University 3400 North Charles Street Baltimore, MD 21218-2685 U.S.A.

Office Telephone: Lab Telephone:  Department Fax:  Email:

410.516.5206 410.516.5207 410.516.5213 person at jhu dot edu where person is schleif

Office- Mudd 239  Lab- Mudd 245

RESEARCH INTERESTS

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.

products by binding to the araO2 and araI1 half-sites and forming a DNA loop that blocks access of RNA polymerase to the pC and pBAD promoters. Upon the addition of arabinose, AraC ceases looping and binds instead to the adjacent half-sites, araI1 and araI2, where it and the cyclic AMP binding protein, CAP, both help RNA polymerase to bind to the pBAD promoter and speed the formation of open complex by RNA polymerase, thereby stimulating the synthesis of the AraB, AraA, and AraD gene products 100- to 500-fold. 

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. 

• Mutational Analysis of Residue Roles in AraC Function, J. Ross, U. Gryczynski, R. Schleif, J. Mol. Biol. 328, 85-93 (2003).
• Modeling and Studying Proteins with Molecular Dynamics, Robert Schleif, Methods in Enzymology 338, Ed. L. Brand, M. Johnson, Elsevier Academic Press, San Diego (2004). 
• Biochemical and Physiological Properties of the DNA Binding Domain of AraC Protein, A. Timmes, M. Rodgers, Robert Schleif, J. Mol. Bio. 340, 731-738 (2004).
• A Portable Allosteric Mechanism, U. Gryczynski, R. Schleif, Proteins, 57, 9-11 (2004).
• Arm-Domain Interactions Can Provide High Binding Cooperativity, R. Schleif, C. Wolberger, Protein Science 13, 2829-2831 (2004).
• Building Family Traditions, R. Schleif, Mol. Micro. 53, 355-356 (2004).
• Specific Interactions by the N-terminal Arm Inhibit Self-association of the AraC Dimerization Domain, John E. Weldon, Robert F. Schleif, Protein Science 15, 2828 – 2835 (2006).
• Structure and Properties of a Truely Apo Form of AraC Dimerization Domain John E. Weldon, Michael E. Rodgers, Christopher Larkin, and Robert F. Schleif , Proteins 66, 646-654 (2007).
  

• Undergraduates
• YinFei Xu
• Neil Neumann
• Shishir Sharma
  
• Graduate Students 
• Victoria Hargreaves, email:  person at jhu dot edu where person is vhargreaves
  
• Jennifer Seedorff
• Katie Frato, email: person at jhu dot edu where person is kfrato
• Stephanie Dirla, email:  person at jhu dot edu where person is sdirla1
  
• Research Scientist: 
• Michael Rodgers, email: person at jhu dot edu where person is rodgers