Title:
Creating a library of genetically encoded heme sensors with varying binding affinities
Creating a library of genetically encoded heme sensors with varying binding affinities
Author(s)
Ashworth, Jessie D
Advisor(s)
Reddi, Amit R.
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Abstract
Heme is an important biological metallo-cofactor that also works as a signaling molecule in cells. Despite its importance, heme trafficking and mobilization in the cell are currently not well understood, in part due to the limited ability to visualize and quantify heme in vivo. This inconvenience has recently been overcome by the development of genetically encoded heme sensors by the Reddi lab. Using these sensors, the Reddi lab has identified new heme trafficking factors and probed the spatio-temporal dynamics of heme mobilization. While the heme sensors are powerful tools for imaging and quantifying heme, improvements could be implemented that would allow for greater utility than is currently available due to particular limitations in the prototype sensors. A current limitation in heme sensing is that the heme dissociation constants of the prototype sensors span a limited range. The high affinity heme sensor, HS1 (KdII ~ 10 pM and KdIII = 9 nM) is quantitatively saturated in all sub-cellular compartments, including the cytosol, nucleus, and mitochondria, making it unsuitable for heme monitoring. The low affinity sensor, HS1-M7A (KdII = 20 nM and KdIII = 2 uM) is ~20-50% saturated with heme in the cytosol, but ~ 0 % saturated in the nucleus and mitochondria, making it unsuitable for heme monitoring in these compartments. In an effort to broaden the utility of the heme sensors, we report herein an expanded library of sensors engineered to span a wide-array of heme affinities. Heme binding residues were mutated to Ala, Cys, His, Lys, Met, Ser and Tyr to generate a panel of 47 new mutants which were found to be between 0 and 100% saturated when expressed in the yeast cytosol. These new tools will enable unprecedented access to cellular heme pools for probing heme homeostasis in health and disease.
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Date Issued
2016-12-09
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