Section: Rotations

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Scot Wolfe, Ph.D.

Academic Role: Associate Professor

Faculty Appointment(s) In:
   Biochemistry and Molecular Pharmacology
   Program in Gene Function and Expression

Other Affiliation(s):
   Interdisciplinary Graduate Program

Potential Rotation Projects

Selection of dimeric zinc finger proteins with novel DNA-binding specificity

Goal: Create Dimeric zinc finger proteins that can recognize target sequences in certain yeast, mouse and human genes.

The DNA-binding specificity of a dimeric zinc finger chimera - created using a combination of structure-based design and phage display - will be altered to recognize new sequences that occur in various genes.  A bacterial two-hybrid system will be used to facilitate the selection of these proteins. The resulting proteins will be examined in vitro with regards to specificity and affinity, and they will then be tested in vivo to determine their ability to alter gene expression.  This project will provide training in a cutting-edge selection methodology and experience with gel-shift assays.  Depending upon the timeframe, it could lead to cell culture experience, and transcriptional profiling to examine the specificity of these proteins in vivo.

Selection of dimeric zinc finger proteins that preferentially heterodimerize

Goal: Define the minimal dimerization element required for cooperative DNA recognition and optimize this sequence to create an obligate heterodimer.

Recently I created a dimeric zinc finger protein using a combination of structure-based design and phage display.  This protein is a fusion between the leucine zipper of GCN4 and the zinc fingers of zif-268.  The junction between these two domains was optimized by phage display in the context of a homodimer.  Currently this system needs to undergo further analysis and optimization.  The dimerization potential of the leucine zipper needs to be attenuated, and also optimized to prefer heterodimerization over homodimerization.  This will involve truncation of the leucine zipper to find the minimal element necessary for cooperative DNA recognition.  Subsequently, this region must be optimized to preferentially heterodimerize with another partner over the homodimerization with another copy of itself.  Two methods of selection will be explored to achieve this goal: phage display and a bacterial two-hybrid system. The resulting proteins will be examined in vitro and in vivo with regards to specificity.  This project will provide training in a cutting-edge selection methodology and depending upon the timeframe, it could lead to crystallization trials of the selected proteins to facilitate structural studies by X-ray crystallography.  (The structure of the homodimer has been solved bound to DNA at a 1.5Å resolution.)

Selection of dimeric zinc finger proteins that bind DNA in a drug-dependent manner

Goal: Create an artificial transcription factor that binds DNA only in the presence/absence of a drug.

A drug regulatable transcription factor that could be targeted to any gene of interest would provide a powerful new tool for researchers studying gene function in vivo.  Building upon the dimeric zinc finger protein system described above, a drug dependent dimerization element will be substituted for the leucine zippers.  This will involve examining the compatibility of structures of drug-based dimerization systems with the structure of the dimeric zinc finger protein.  Based on computer modeling, prototypes of various fusions will be constructed and tested.  Promising leads will then be optimized by phage display or using a bacterial two-hybrid system. The resulting proteins will be examined in vitro and in vivo with regards to drug dependent-DNA recognition.  This project will provide training in basic computer modeling of proteins and in a cutting-edge selection methodology.  Depending upon the timeframe, it could lead to cell culture experience and transcriptional profiling when examining the function of these proteins in vivo.

Office: LRB 619
Phone: 508-856-3953
E-mail: Scot.Wolfe@umassmed.edu
Keywords: Protein-DNA recognition, Gene Therapy, Structural Biology, Biochemistry, Gene Regulation

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