Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function.

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. GFP homodimers demonstrate synergistic behavior with the dimer being brighter than the sum of the monomers. The homodimer structure reveals the chromophores are close with favorable transition dipole alignments and a highly solvated interface. Heterodimerization (GFP with Venus) results in a complex with ≈87% FRET efficiency, significantly below the 99.7% efficiency predicted. A similar efficiency is observed when the wild-type FPs are fused to a naturally occurring protein-protein interface system. GFP complexation with mCherry results in loss of mCherry fluorescence. Thus, simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction.

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...

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the functionally important cap region is significantly different to EstN7's closest relatives, forming a bridge-like structure with reduced helical content providing greater access to the active site through more than one substrate access tunnel. However, dynamics do not appear to play a major role in cold adaption. Molecular dynamics at different temperatures, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are important for of EstN7's cold adaptation rather than changes in dynamics.

The previously published version of this Article contained a consequential error in the fourth sentence of the "Discussion", which incorrectly read 'The use of the azF-SCO crosslink proved important as classical disulfide crosslinking did improve protein function'. The correct version states 'The use of the azF-SCO crosslink proved important as classical disulfide crosslinking did not improve protein function'-the word 'not' has been added as the 14th word. This has been corrected in both the PDF and HTML versions of the Article.

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus 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 HblL1 component and the elucidation of its structure to 1.36 Å. Like HblB, it is a member of the alpha-helical pore-forming toxin family. In comparison to other members of this group, it has an extended hydrophobic beta tongue region that may be involved in pore formation. Molecular docking was used to predict possible interactions between HblL1 and HblB, and suggests a head to tail dimer might form, burying the HblL1 beta tongue region.

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1, which has an apparent melting temperature of 26°C. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. However, dynamics do not appear to play a major role in cold adaption. Comparison of B-factors with the closest related mesophilic and thermophilic esterases suggests there is little difference in dynamics with the catalytically important N-terminal cap comprising the main dynamic element. Molecular dynamics, rigidity analysis, normal mode analysis and geometric simulations of motion confirm the flexibility of the cap region but suggest that the rest of the protein is largely rigid. Rigidity analysis indicates the distribution of hydrophobic tethers is appropriate to colder conditions, where the hydrophobic effect is weaker than in mesophilic conditions due to reduced water entropy. The conformation of the cap region is significantly different to EstN7′s closest relatives, forming a bridge-like structure with reduced helical content providing more than one access tunnel through to the active site. Thus, it is likely that increased substrate accessibility and tolerance to changes in water entropy are the main drivers of EstN7′s cold adaptation rather than changes in dynamics.

Klebsiella pneumoniae is an adaptable pathogen that forms biofilms on a variety of surfaces. This study's objective was to identify the presence of fimbrial genes (types 1 and 3) in K. pneumoniae strains isolated from various clinical sources based on their antibiotic resistance and ability to form biofilms. According to identification utilizing the vitek 2 technology and confirmation by molecular identification targeting the 16S rRNA gene with a particular primer, forty isolates were identified from clinical specimens. The vitek 2 compact system was utilized to evaluate the antibiotic susceptibility of all the isolates. The findings revealed a range of resistance percentages, including 52.5% for Penicillin, 40.5% for Trimethoprim/Sulfamethoxazole, 34.5% for Cephalosporins, 6.25 % for Fluoroquinolones, and 2.5% for each of Carbapenem, Aminoglycoside, Tetracycline, and Nitrofurantoin. The 96-well microtiter plate technique was utilized to generate biofilms. The results demonstrated that all 40 Klebsiella pneumoniae isolates (100%) produced potent biofilms. In order to identify the genes involved in biofilm formation (fimh & mrkd) and the genes responsible for adhesin in type 1& type 3 fimbriae using traditional PCR method, eleven isolates were chosen for molecular analysis that are powerful biofilm makers and MDR.