Andy Dixon, PhD

 

Research Projects

A critical aspect of shuttling proteins to an alternate intracellular location is the capture of the target protein. This capture ideally will involve high affinity and specificity. I am undertaking two approaches to capture Bcr-Abl: rationally-designed coiled-coils and single domain antibodies. I am also working on development of a new assay (NTA) to detect protein-protein interactions in vivo.

Rationally Designed Coiled-Coils

Bcr-Abl is found predominantly as homo-oligomers, and the oligomerization is achieved through a coiled-coil domain at the N-terminus of the protein. Although this coiled-coil domain might be used for capture of Bcr-Abl, it would have no specificity towards Bcr-Abl over another coiled-coil domain, and thermodynamically oligomers of the isolated coiled-coil might be favored over the oligomerization with Bcr-Abl. To circumvent this specificity problem, and to obtain a higher affinity interaction, I am working on designing mutant coiled-coils that incorporate charged residues at key positions in attempt to introduce charge-charge repulsion between two mutant coiled-coil domains and increase the number of salt bridges between the mutant and Bcr-Abl coiled-coil domains. This is a collaborative project with Dr. Thomas Cheatham (Medicinal Chemistry, University of Utah) who is an expert on coiled-coils and molecular dynamic simulations.

Single Domain Antibodies (iDabs)

Single domain antibodies (iDabs) consist of one variable domain (heavy or light chain) and are small (≈13kDa), stable (including inside of cells), and retain the high affinity and specificity common to antibodies. Dr. Terence Rabbitts and Dr. Tomoyuki Tanaka at the Leeds Institute of Molecular Medicine (LIMM) are the pioneers of iDabs and have developed a technique termed intracellular antibody capture (IAC) for the isolation of iDabs that bind to a given target. Dr. Rabbitts was generous enough to allow me to come to his laboratory in Leeds, UK where I was trained in the IAC procedure. I am working to generate iDabs that bind Bcr-Abl at both the actin binding and DH/PH domains with the aim of competing with other interactions that anchor Bcr-Abl to the cytoplasm.

Nuclear Translocation Assay (NTA)

Another application of the protein switch technology is to assay protein-protein interactions under cellular conditions. The controlled nuclear translocation of the protein switch can be turned into a mechanism to study protein interactions by fusing one protein of interest to the protein switch and subsequently monitoring the nuclear translocation of a second protein of interest. As the second protein should not otherwise translocate into the nucleus, its nuclear translocation is indicative of an interaction with the protein fused to the protein switch. This methodology, termed the nuclear translocation assay (NTA), has been demonstrated through fluorescence microscopy of EGFP and dsRed fusion constructs. Future directions will be to develop a system allowing easier quantification of the cytoplasmic/nuclear localization of the two proteins providing a more high-throughput assay.