C. Bamdad, THE USE OF VARIABLE-DENSITY SELF-ASSEMBLED MONOLAYERS TO PROBE THE STRUCTURE OF A TARGET MOLECULE, Biophysical journal, 75(4), 1998, pp. 1989-1996
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.