Faculty of 1000 Review

Generation of longer emission wavelength red fluorescent proteins using computationally designed libraries.
RA Chica, MM Moore, BD Allen, SL Mayo
Proc Natl Acad Sci U S A 2010 Nov 23 107 47:20257-62

Selected and evaluated by Brian Kuhlman.

Evaluation details:

Must Read [8]



Directed Molecular Evolution,Experimental Biophysical Methods,Theory & Simulation,Protein Folding

We found this article interesting because, to our knowledge, it is the first demonstration of screening computer-based, focused protein libraries designed explicitly to achieve a specific function (red-shifted emission wavelength). Computational protein design and high-throughput screening of large protein libraries have been merged in several previous studies {1-3}. Although the screens have identified mutants with the desired function, the computational design was performed to enrich libraries with mutants with better binders, or well-folded mutants. Direct computational optimization for a function such as catalysis or fluorescence has never been performed to design-directed libraries. In this study, Chica and coworkers computationally redesigned the environment of mCherry fluorophore to red-shift the emission wavelength based on three hypotheses. They looked for mutations that stabilized the excited state or destabilized the ground state to achieve a longer emission wavele ngth. They designed libraries with <1,000 members and screened these mutant libraries for red-shifted mutants. Following the initial screen, they combined mutations from different libraries to achieve a 26 nm red-shift in emission wavelength. Crystal structures showed good agreement with the designed models. In the future, it will be interesting to see whether this strategy can be used to construct directed libraries for the de novo design of novel enzymes.

{1} Treynor et al. Proc Natl Acad Sci USA 2007, 104:48-53 [PMID:17179210].
{2} Mena et al. Nat Biotechnol 2006, 24:1569-71 [PMID:17115054].
{3} Hayes et al. Proc Natl Acad Sci USA 2002, 99:15926-31 [PMID:12446841].