InteractiveROSETTA is freely available at https://github.com/schenc3/InteractiveROSETTA/releases and relies upon a separate download of PyRosetta which is available at http://www.pyrosetta.org after obtaining a license (free for academic use).

Leave-one-out green fluorescent protein (LOOn-GFP) is a circularly permuted and truncated GFP lacking the nth β-strand element. LOO7-GFP derived from the wild-type sequence (LOO7-WT) folds and reconstitutes fluorescence upon addition of β-strand 7 (S7) as an exogenous peptide. Computational protein design may be used to modify the sequence of LOO7-GFP to fit a different peptide sequence, while retaining the reconstitution activity. Here we present a computationally designed leave-one-out GFP in which wild-type strand 7 has been replaced by a 12-residue peptide (HA) from the H5 antigenic region of the Thailand strain of H5N1 influenza virus hemagglutinin. The DEEdesign software was used to generate a sequence library with mutations at 13 positions around the peptide, coding for approximately 3 × 105 sequence combinations. The library was coexpressed with the HA peptide in E. coli and colonies were screened for in vivo fluorescence. Glowing colonies were sequenced, and one (LOO7-HA4) with 7 mutations was purified and characterized. LOO7-HA4 folds, fluoresces in vivo and in vitro, and binds HA. However, binding results in a decrease in fluorescence instead of the expected increase, caused by the peptide-induced dissociation of a novel, glowing oligomeric complex instead of the reconstitution of the native structure. Efforts to improve binding and recover reconstitution using in vitro evolution produced colonies that glowed brighter and matured faster. Two of these were characterized. One lost all affinity for the HA peptide but glowed more brightly in the unbound oligomeric state. The other increased in affinity to the HA peptide but still did not reconstitute the fully folded state. Despite failing to fold completely, peptide binding by computational design was observed and was improved by directed evolution. The ratio of HA to S7 binding increased from 0.0 for the wild-type sequence (no binding) to 0.01 after computational design (weak binding) and to 0.48 (comparable binding) after in vitro evolution. The novel oligomeric state is composed of an open barrel.

The autocatalytic maturation of the chromophore in green fluorescent protein (GFP) was thought to require the precise positioning of the side chains surrounding it in the core of the protein, many of which are strongly conserved among homologous fluorescent proteins. In this study, we screened for green fluorescence in an exhaustive set of point mutations of seven residues that make up the chromophore microenvironment, excluding R96 and E222 because mutations at these positions have been previously characterized. Contrary to expectations, nearly all amino acids were tolerated at all seven positions. Only four point mutations knocked out fluorescence entirely. However, chromophore maturation was found to be slower and/or fluorescence reduced in several cases. Selected combinations of mutations showed nonadditive effects, including cooperativity and rescue. The results provide guidelines for the computational engineering of GFPs.

Programs for splitting protein design expressions are available at www.bioinfo.rpi.edu/tools/piecemeal.html CONTACT: bystrc@rpi.edu Supplementary information: Supplementary data are available at Bioinformatics online.