THE USE OF VARIABLE-DENSITY SELF-ASSEMBLED MONOLAYERS TO PROBE THE STRUCTURE OF A TARGET MOLECULE

VP16, a protein encoded by herpes simplex virus, has a well-characteri zed 78 amino acid acidic activation domain. When tethered to DNA, tand em repeats of an eight amino acid motif taken from this region stimula te the transcription of a nearby gene. This work addresses how these m inimal activation motifs interact with a putative target, the general transcription factor TATA box binding protein (TBP), and the biologica l relevance of this mechanism of action. I developed novel biophysical techniques to discriminate among three possible mechanistic models th at describe how reiterated peptide motifs could synergistically effect transcription: 1) the peptide motifs simultaneously bind to quasi-ide ntical sites on TBP, producing a high-affinity bivalent interaction th at holds the general transcription factor near the start site of trans cription; 2) the binding of one recognition motif causes an allosteric effect that enhances the subsequent binding of additional peptide mot ifs; or 3) a high-affinity interaction between the peptide repeats and TBP does occur, but rather than being the result of a ''bivalent'' in teraction, it results from the summation of multiple interactions betw een the target protein and the entire length of the peptide. I generat ed self-assembled monolayers (SAMs) that presented different densities of the activation motif peptide in a two-dimensional array to test fo r avidity effects. Surface plasmon resonance (SPR) was used to measure the amount of target (TBP) binding as a function of the peptide densi ty; a marked increase in avidity above a characteristic, critical pept ide surface density was found. Competitive inhibition experiments were performed to compare the avidity of peptide motifs, tandemly repeated two or four times, and single motifs separated by a flexible linker. Four iterations of the motif, preincubated with TBP, inhibited its bin ding to high-density peptide surfaces similar to 250-fold better than two iterations. Single peptide motifs joined by a flexible amino acid linker inhibited TBP binding to surface peptide nearly as well as four tandem repeats. The results favor mechanistic model 1: reiterated act ivation motifs interact with TBP through a high-affinity interaction t hat is the result of the cooperative effect of single motifs simultane ously binding to separate sites on TBP. This finding is consistent wit h the idea that DNA-bound activation domains trigger the transcription of a nearby gene by tethering the general transcription factor, TBP, near the start site of transcription.