Team:BostonU/Modeling/Dimerization Domain

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Modeling

Dimerization Domains

Experimental Workflow

Dimerization Domains

In our project we explored the three different conditional dimerization domain pairs: FKBP and FRB domains induced by the small molecule Rapalog, ABI and PYL domains induced by the small molecule Abscisic Acid, and CRY2 and CIBN domains induced by blue light stimulus. We looked into three systems because each had its own advantages. We hypothesized that these different domains would give different levels of protein activity control. Additionally, each of these systems is orthogonal, so theoretically multiple conditionally-dimerized proteins could be controlled in the same cell.

FKBP/FRB/Rapalog

This conditional dimerization system is the most widely studied in the literature. The two protein domains are the smallest pair of the three that we investigated, and we hypothesized that smaller domains would be advantageous because they might have the least interference with folding of the independent split halves and overall protein activity when dimerized. In addition, FKBP and FRB bind very tightly in the presence of rapalog, which means that only a small amount of inducer needs to be administered to induce dimerization. One disadvantage of this system is that FKBP and FRB do not easily come apart after dimerization is induced. This could be troublesome for applications in which protein activity needs to be rapidly modulated. Additionally, Rapalog does have some off target effects in mammalian cells, which could be potentially troublesome for in vivo applications¹.

ABI/PYL/Abscisic Acid

This conditional dimerization system induced by Abscisic Acid (ABA) is native to plant cells. For this reason, the system exhibits very little off-target effects in mammalian systems. These domains are larger in size compared to FKBP and FRB, which may interfere protein folding and dimerized activity. ABI and PYL also bind less tightly than FKBP and FRB in the presence of the corresponding inducer, which means more inducer may need to be administered to get the same level of dimerization. However, the dimerization reaction can be reversed by washing cells with media that does not contain ABA².

CRY2/CIBN/Blue Light

This conditional dimerization system is particularly exciting because it is not induced by a small molecule, but rather by blue light. While small molecules are free to diffuse, light can have a high spatial resolution. This property is useful in inducing protein activity in specific in vivo areas. This dimerization system is also very fast; in the presence of blue light the domains can come together on the order of milliseconds and can separate in minutes. However, CRY2 and CIBN are very large compared to the above domains induced by small molecules, which may possibly lead to decreased function in dimerized proteins³.

Citations

1. Ho, Steffan N. et al., “Dimeric ligands define a role for transcriptional activation domains in reinitiation”, Nature 1996.
2. Liang, Fu-Sen, Ho, Wen Qi, Crabtree, Gerald R., “Engineering the ABA Plant Stress Pathway for Regulation of Induced Proximity”, Science Signaling, 2011.
3. Kennedy Matthew J. et al., “Rapid blue-light–mediated induction of protein interactions in living cells”, Clinical Nature Methods, 2010.