Enhanced Green Fluorescent Protein (EGFP) is one of the most widely used engineered variants of the original wild-type Green Fluorescent Protein. Here, we report the high resolution (1.35 Å) structure of EGFP crystallised in its untagged sequence form that reveals the combined impact of the F64L and S65T, that give rise to improved folding and spectral characteristics. The overall structure of EGFP is very similar to wt GFP, forming the classical β-barrel fold with the chromophore containing helix running through the core of the structure. Replacement of Phe64 with Leu in EGFP results in subtle rearrangement of hydrophobic core packing close to the chromophore including the reduction in surface exposure of two hydrophobic residues. Replacement of Ser65 with Thr has a significant impact on the local hydrogen bond network in the vicinity of the chromophore. Detailed analysis of electron density reveals that several residues close to the chromophore occupy at least two distinct conformations. This includes Glu222 that defines the charged state on the chromophore, with the two conformations having slightly different effects on the hydrogen bond network surrounding the chromophore. Hence, the reported high-resolution structure of EGFP has provided a long overdue molecular description of one of the most important fluorescent protein variants currently in general use.

Synthetic Biology is the ‘Engineering of Biology’ – it aims to use a forward-engineering design cycle based on specifications, modelling, analysis, experimental implementation, testing and validation to modify natural or design new, synthetic biology systems so that they behave in a predictable fashion. Motivated by the need for truly plug-and-play synthetic biological components, we present a comprehensive review of ways in which the various parts of a biological system can be modified systematically. In particular, we review the list of ‘dials’ that are available to the designer and discuss how they can be modelled, tuned and implemented. The dials are categorized according to whether they operate at the global, transcriptional, translational or post-translational level and the resolution that they operate at. We end this review with a discussion on the relative advantages and disadvantages of some dials over others.

SummaryAltering a protein’s backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate residue deletion across the breadth of the protein, particularly within short and long loops, helical elements, and at the termini of strands. A variant with G4 removed from a helix (EGFPG4Δ) conferred significantly higher cellular fluorescence. Folding analysis revealed that EGFPG4Δ retained more structure upon unfolding and refolded with almost 100% efficiency but at the expense of thermodynamic stability. The EGFPG4Δ structure revealed that G4 deletion caused a beneficial helical registry shift resulting in a new polar interaction network, which potentially stabilizes a cis proline peptide bond and links secondary structure elements. Thus, deletion mutations and registry shifts can enhance proteins through structural rearrangements not possible by substitution mutations alone.

The construction of useful functional biomolecular components not currently part of the natural repertoire is central to synthetic biology. A new light-capturing ultra-high-efficiency energy transfer protein scaffold has been constructed by coupling the chromophore centers of two normally unrelated proteins: the autofluorescent protein enhanced green fluorescent protein (EGFP) and the heme-binding electron transfer protein cytochrome b(562) (cyt b(562)). Using a combinatorial domain insertion strategy, a variant was isolated in which resonance energy transfer from the donor EGFP to the acceptor cyt b(562) was close to 100% as evident by virtually full fluorescence quenching on heme binding. The fluorescence signal of the variant was also sensitive to the reactive oxygen species H(2)O(2), with high signal gain observed due to the release of heme. The structure of oxidized holoprotein, determined to 2.75 Å resolution, revealed that the two domains were arranged side-by-side in a V-shape conformation, generating an interchromophore distance of ~17 Å (14 Å edge-to-edge). Critical to domain arrangement is the formation of a molecular pivot point between the two domains as a result of different linker sequence lengths at each domain junction and formation of a predominantly polar interdomain interaction surface. The retrospective structural analysis has provided an explanation for the basis of the observed highly efficient energy transfer through chromophore arrangement in the directly evolved protein scaffold and provides an insight into the molecular principles by which to design new proteins with coupled functions.

Ionic liquids offer exciting possibilities for biocatalysis as solvent properties provide rare opportunities for customizable, energy-efficient bioprocessing. Unfortunately, proteins and enzymes are generally unstable in ionic liquids and several attempts have been made to explain why; however, a comprehensive understanding of the ionic liquid–protein interactions remains elusive. Here, we present an analytical framework (circular dichroism (CD), fluorescence, ultraviolet-visible (UV/Vis) and nuclear magnetic resonance (NMR) spectroscopies, and small-angle X-ray scattering (SAXS)) to probe the interactions, structure, and stability of a model protein (green fluorescent protein (GFP)) in a range (acetate, chloride, triflate) of pyrrolidinium and imidazolium salts. We demonstrate that measuring protein stability requires a similar holistic analytical framework, as opposed to single-technique assessments that provide misleading conclusions. We reveal information on site-specific ionic liquid–protein interactions, revealing that triflate (the least interacting anion) induces a contraction in the protein size that reduces the barrier to unfolding. Robust frameworks such as this are critical to advancing non-aqueous biocatalysis and avoiding pitfalls associated with single-technique investigations.