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Published online on December 19, 2006, 10.1073/pnas.0609647103
PNAS | January 2, 2007 | vol. 104 | no. 1 | 48-53


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BIOLOGICAL SCIENCES / BIOCHEMISTRY
Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function

Thomas P. Treynor*,{dagger}, Christina L. Vizcarra{ddagger}, Daniel Nedelcu*, and Stephen L. Mayo*,{dagger},§

Divisions of *Biology and Chemistry and {ddagger}Chemical Engineering and {dagger}Howard Hughes Medical Institute, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125

Contributed by Stephen L. Mayo, October 31, 2006 (received for review August 11, 2006)

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.

GFP | library design | protein design | protein engineering | high-throughput screening

Author contributions: T.P.T. and S.L.M. designed research; T.P.T., C.L.V., and D.N. performed research; T.P.T. contributed new reagents/analytic tools; T.P.T. and C.L.V. analyzed data; and T.P.T. wrote the paper.

The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0609647103/DC1.

§To whom correspondence should be addressed. E-mail: steve{at}mayo.caltech.edu

© 2007 by The National Academy of Sciences of the USA


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