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Engineering Monomerization of Red Fluorescent Proteins Through Computational Design

Wannier, Timothy and Moore, Matthew and Mayo, Stephen (2012) Engineering Monomerization of Red Fluorescent Proteins Through Computational Design. Protein Science, 21 (S1). pp. 144-145. ISSN 0961-8368. http://resolver.caltech.edu/CaltechAUTHORS:20120828-072233569

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Abstract

Fluorescent proteins (FPs) have, over the last two decades, revolutionized the way biology and biochemistry are studied. However, the obligate oligomerization of many naturally occurring fluorescent proteins has hampered their usefulness as biological markers. Oligomerization of a fusion tag can impair the accuracy of characterizing a target protein, artificially aggregating its target, altering diffusion rates, and causing problems in target transport and trafficking. A monomeric fluorescent protein avoids these problems. In this vein, DsRed, a well-studied red fluorescent protein (RFP), was successfully engineered through directed evolution into monomeric mCherry by Roger Tsien and coworkers. This was the first instance of a monomeric RFP and filled a void in the color spectrum for monomeric molecular labels. DsRed is only one of hundreds of naturally occurring fluorescent proteins, many diverging significantly in fluorescent properties such as absorbance and emission spectra or quantum yields. Repeating the directed evolution process in other proteins would be a long and laborious endeavor, and therefore, a new method is desired for engineering monomerization into fluorescent proteins. We have devised a process through which we can engineer RFP monomers in silica via computational protein design (CPD) of surface positions at the oligomerization interfaces. To validate the method, we first engineered DsRed that had been modified to include mutations made to the core of the protein during its directed evolution to mCherry. CPD predicted sequence variants from which a 96-member library was constructed; a 24-member randomly generated library was also used as a control. The random library allowed hydrophilic residues, but weighted them based on their frequency of occurrence on the surface of bacterial cytoplasmic proteins. The design library maintained fluorescence in 96.8% of cases, whereas only 9.1% of the random library did so. These results demonstrate the power of our design method. The designed proteins were confirmed to be monomeric by gel filtration, homoFRET, and analytical ultracentrifugation. As expected from the mutations to the core of the protein, the mutants maintained the spectroscopic properties ofmCherry and not of OsRed. We hope to repeat these results in other fluorescent proteins that have yet to be monomerized such as HcRed and AQ143, which are both significantly red-shifted from DsRed.


Item Type:Article
Record Number:CaltechAUTHORS:20120828-072233569
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:20120828-072233569
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:33586
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:28 Aug 2012 20:36
Last Modified:23 Aug 2016 10:17

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