Green fluorescent protein (GFP) and GFP-like fluorescent proteins owe their photophysical properties to an autocatalytically formed intrinsic chromophore. According to quantum mechanical calculations, the excited state of chromophore model systems has significant dihedral freedom, which may lead to fluorescence quenching intersystem crossing. Molecular dynamics simulations with freely rotating chromophoric dihedrals were performed on green, yellow, and blue fluorescent proteins in order to model the dihedral freedom available to the chromophore in the excited state. Most current theories suggest that a restriction in the rotational freedom of the fluorescent protein chromophore will lead to an increase in fluorescence brightness and/or quantum yield. According to our calculations, the dihedral freedom of the systems studied (BFP > A5 > YFP > GFP) increases in the inverse order to the quantum yield. In all simulations, the chromophore undergoes a negatively correlated hula twist (also known as a bottom hula twist mechanism).

Green Fluorescent Proteins (GFP) and GFP-like proteins all undergo an autocatalytic posttranslational modification to form a centrally located chromophore. Structural analyses of all the GFP and GFP-like proteins in the protein databank were undertaken to determine the role of the tightturn, broken hydrogen bonding, Gly67, Glu222 and Arg96 in the biosynthesis of the imidazolone group from 65SYG67. The analysis was supplemented by computational generation of the conformation adopted by uncyclized wild-type GFP. The data analysis suggests that Arg96 interacts with the Tyr66 carbonyl, stabilizing the reduced enolate intermediate that is required for cyclization; the carboxylate of Glu 222 acts as a base facilitating, through a network of two waters, the abstraction of a hydrogen from the α-carbon of Tyr66; a tight-turn conformation is required for autocatalytic cyclization. This conformation is responsible for a partial reduction in the hydrogen bonding network around the chromophore-forming region of the immature protein.

The Notch signaling pathway plays an important role during development in animals from worms to humans and pathway components are required for the differentiation of many different cell types. In Drosophila, Su(H) dependent Notch activation up-regulates transcription of the Enhancer of split-Complex (E(spl)-C). The E(spl) genes are known to function during neurogenesis, although expression and genetics studies suggest that they also play roles in the development of other tissues. The majority of the E(spl) genes contain upstream binding sites for Su(H), proneural proteins, and E(spl) bHLH proteins resulting in overlapping expression patterns during embryonic development. However, their expression patterns are quite distinct during later embryonic stages and in larval imaginal discs. In order to characterize expression patterns of the E(spl) genes during development and determine potential mechanisms through which expression is controlled, we examined the expression levels and patterns of the E(spl) genes in the midgut during metamorphosis. Quantitative Reverse Transcriptase-PCR and X-Gal staining results show that the genes have different levels and patterns of expression in the developing midgut. Two ancestral E(spl) genes, malpha and mbeta, are highly expressed and increase significantly at puparium formation, whereas another gene, mgamma, is expressed at low levels and decreases in expression at puparium formation. We also show that mbeta is expressed in cells throughout the midgut, while mgamma is expressed in two small regions. These results provide further evidence that the E(spl) genes function during midgut development and that they are regulated by different factors.