The deletion of amino acids is one of the evolutionary mechanisms by which nature adapts the function of proteins. A simple method has been developed that mimics this event in vitro by introducing a deletion of exactly three nucleotides at random positions in a target gene. The method involved the engineering of the mini-Mu transposon to introduce a recognition sequence for the restriction enzyme MlyI. The new transposon, MuDel, was capable of efficient insertion into a target DNA sequence. To determine the efficacy of the method, the bla gene that encodes the TEM-1 β-lactamase was used as the target and a small library containing 22 different sequence variants was created. Of these 22 variants, 8 were identified that conferred resistance to ampicillin on Escherichia coli . Each of the TEM-1 variants possessed a distinct ampicillin minimum inhibitory concentration, ranging from 500 to >10 000 μg/ml. Sequence analysis revealed that active TEM-1 variants contained deletions not just in loops but also helices, and included regions known to be involved in catalysis, antibiotic resistance and inhibitor binding. This new technology is transferable to most genes, permitting an extensive analysis of deletion mutations on protein function.

GFP and a FRET compatible dye were used to assess the influence of genetically encoded aryl azide positioning on Click chemistry-based protein conjugation. While modification efficiency of the sampled mutants using a strain promoted reaction varied by as much as ∼10 fold, there was no simple correlation with accessibility of the aryl azide on GFP's surface. One labeled GFP mutant (Gln204AzPhe) exhibited high efficiency FRET (∼90%) and an unprecedented pseudo-Stokes shift of 126 nm.

We demonstrate an approach that allows attachment of single-stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multicomponent assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemistry was used to attach exactly one ssDNA at precisely defined residues. By choosing an appropriate residue, ssDNA conjugation had minimal impact on protein function, even when attached close to active sites. The protein-ssDNA conjugates were used to (i) assemble double-stranded DNA systems with optimal communication (energy transfer) between normally separate groups and (ii) generate multicomponent systems on DNA origami tiles, including those with enhanced enzyme activity when bound to the tile. Our approach allows any potential protein to be simply engineered to attach ssDNA or related biomolecules, creating conjugates for designed and highly precise multiprotein nanoscale assembly with tailored functionality.

Glycosylation is the most prevalent protein post‐translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure–function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme horseradish peroxidase (HRP), which in turn increases its turnover and stability. Circular dichroism spectroscopy revealed that glycosylation increased holo‐HRP's thermal stability and promoted significant helical structure in the absence of haem (apo‐HRP). Glycosylation also resulted in a 10‐fold increase in enzymatic turnover towards o‐phenylenediamine dihydrochloride when compared to its nonglycosylated form. Utilising a naturally occurring site‐specific probe of active site flexibility (Trp117) in combination with red‐edge excitation shift fluorescence spectroscopy, we found that glycosylation significantly rigidified the enzyme. In silico simulations confirmed that glycosylation largely decreased protein backbone flexibility, especially in regions close to the active site and the substrate access channel. Thus, our data show that glycosylation does not just have a passive effect on HRP stability but can exert long‐range effects that mediate the ‘native’ enzyme's activity and stability through changes in inherent dynamics.