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Published January 2, 2007 | Supplemental Material + Published
Journal Article Open

Computationally designed libraries of fluorescent proteins evaluated by preservation and diversity of function


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.

Additional Information

© 2007 by The National Academy of Sciences of the USA Contributed by Stephen L. Mayo, October 31, 2006 (received for review August 11, 2006) Published online before print December 19, 2006, 10.1073/pnas.0609647103 We thank Patrick Daugherty (University of California, Santa Barbara, CA) for providing many of the primers used to assemble GFP-S65T and a pBAD-derived vector engineered with SfiI recognition sequences; Christina Smolke for the use of her plate reader; Marco Mena, Michelle Meyer, and Frances Arnold for advice in designing this project; and Marie Ary for useful comments on the manuscript. This research was supported by the Howard Hughes Medical Institute and the Army Research Office. T.P.T. was supported by National Institutes of Health Grant F32-GM07438. C.L.V. was supported by a National Science Foundation Graduate Research Fellowship. 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.

Attached Files

Published - TREpnas07.pdf

Supplemental Material - 09647Fig5.pdf

Supplemental Material - 09647Fig6.pdf

Supplemental Material - 09647Fig7.pdf

Supplemental Material - 09647Fig8.pdf

Supplemental Material - 09647Fig9.pdf


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