Selection of protein core stability using combinatorial peptide microarrays

BIOL 83

Nathan B. Mahany, nmahany@uvm.edu, Chemistry Department, University of Vermont, 82 University Place, Cook Physical Science Bldg, Burlington, VT 05405 and Martin A. Case, martin.case@uvm.edu, Department of Chemistry, University of Vermont, 82 University Place, Burlington, VT 05405.
The ability to design and synthesize optimally stable proteins could lead to successful protein therapeutics. Such proteins must evade the intracellular degradative machinery, and to this end we are seeking proteins that have negligible unfolded content under biological conditions. We are developing microarrays in which multiple peptide modules, each with a predetermined secondary structural preference, can self-associate to form protein tertiary structures. The stability of a particular assembly is reported using fluorescent resonance energy transfer (FRET). A covalently tethered polypeptide subunit is presented at a specific location on a microarray. Robotic spotting adds two more subunits, one augmented by a FRET donor, the other by a FRET acceptor. A stable assembly is indicated by emission from the acceptor moiety after washing the microarray plate. Stability can be quantitated by repeating the emission measurement using increasing concentrations of denaturant. Proof of concept is demonstrated using the complementary leucine zipper domains of the fos/jun transcription factor pair.
 

Frontiers in Chemical Biology
5:00 PM-7:00 PM, Sunday, August 19, 2007 BCEC -- Exhibit Hall - B2, Poster

Division of Biological Chemistry

The 234th ACS National Meeting, Boston, MA, August 19-23, 2007