Linking the functions of unrelated proteins using a novel directed evolution domain insertion method

We have successfully developed a new directed evolution method for generating integral protein fusions comprising of one domain inserted within another. Creating two connections between the insert and accepting parent domain can result in the inter-dependence of the separate protein activities, thus providing a general strategy for constructing molecular switches. Using an engineered transposon termed MuDel, contiguous trinucleotide sequences were removed at random positions from the bla gene encoding TEM-1 β-lactamase. The deleted trinucleotide sequence was then replaced by a DNA cassette encoding cytochrome b562 with differing linking sequences at each terminus and sampling all three reading frames. The result was a variety of chimeric genes encoding novel integral fusion proteins that retained TEM-1 activity. While most of the tolerated insertions were observed in loops, several also occurred close to the termini of α-helices and β-strands. Several variants conferred a switching phenotype on Escherichia coli, with bacterial tolerance to ampicillin being dependent on the presence of haem in the growth medium. The magnitude of the switching phenotype ranged from 4- to 128-fold depending on the insertion position within TEM-1 and the linker sequences that join the two domains

A Predictive Model of Intein Insertion Site for Use in the Engineering of Molecular Switches

Inteins are intervening protein domains with self-splicing ability that can be used as molecular switches to control activity of their host protein. Successfully engineering an intein into a host protein requires identifying an insertion site that permits intein insertion and splicing while allowing for proper folding of the mature protein post-splicing. By analyzing sequence and structure based properties of native intein insertion sites we have identified four features that showed significant correlation with the location of the intein insertion sites, and therefore may be useful in predicting insertion sites in other proteins that provide native-like intein function. Three of these properties, the distance to the active site and dimer interface site, the SVM score of the splice site cassette, and the sequence conservation of the site showed statistically significant correlation and strong predictive power, with area under the curve (AUC) values of 0.79, 0.76, and 0.73 respectively, while the distance to secondary structure/loop junction showed significance but with less predictive power (AUC of 0.54). In a case study of 20 insertion sites in the XynB xylanase, two features of native insertion sites showed correlation with the splice sites and demonstrated predictive value in selecting non-native splice sites. Structural modeling of intein insertions at two sites highlighted the role that the insertion site location could play on the ability of the intein to modulate activity of the host protein. These findings can be used to enrich the selection of insertion sites capable of supporting intein splicing and hosting an intein switch

Domain insertions in protein structures

Domains are the structural, functional or evolutionary units of proteins. Proteins can comprise a single domain or a combination of domains. In multi-domain proteins, the domains almost always occur end-to-end, i.e., one domain follows the C-terminal end of another domain. However, there are exceptions to this common pattern, where multi-domain proteins are formed by insertion of one domain (insert) into another domain (parent). Here, we provide a quantitative description of known insertions in the Protein Data Bank (PDB). We found that 9% of domain combinations observed in non-redundant PDB are insertions. Although 90% of all insertions involve only one insert, proteins can clearly have multiple (nested, two-domain and three-domain) inserts. We also observed correlations between the structure and function of a domain and its tendency to be found as a parent or an insert. There is a bias in insert position towards the C terminus of parents. We observed that the atomic distance between the N and C terminus of an insert is significantly smaller when compared to the N-to-C distance in a parent context or a single domain context. Insertions are found always to occur in loop regions of parent domains. Our observations regarding the relationship between domain insertions and the structure, function and evolution of proteins have implications for protein engineering

Structural Basis for Efficient Chromophore Communication and Energy Transfer in a Constructed Didomain Protein Scaffold

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