Fluorescent Proteins: Crystallization, Structural Determination, and Nonnatural Amino Acid Incorporation.

Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge has in turn led to the construction of novel variants with new and improved properties.

Stalling chromophore synthesis of the fluorescent protein Venus reveals the molecular basis of the final oxidation step.

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presence of O located above a dehydrated enolate form of the imidazolone ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an "open" conformation so forming a channel that allows O access to the immature chromophore.

The Crystal Structure of HblL.

The Hbl toxin is a three-component haemolytic complex produced by strains and implicated as a cause of diarrhoea in food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL component and the elucidation of its structure to 1.

Association of Fluorescent Protein Pairs and Its Significant Impact on Fluorescence and Energy Transfer.

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio-orthogonal covalent linkages. In each tested case dimerization changes inherent fluorescence, including FRET.