Directed Evolution of Sortase Activity and Specificity

Nature employs complex networks of protein-tailoring enzymes to effect the post-translational modification of proteins in vivo. By comparison, modern chemical methods rely upon either nonspecific labeling techniques or upon the genetic incorporation of bioorthogonal handles. To develop truly robust bioconjugates it is necessary to develop methods which possess the exquisite activity and specificity observed in biological catalysts. One attractive strategy to achieve this is the engineering of protein-tailoring enzymes possessing user-defined specificity and high catalytic efficiency.Chemistry and Chemical Biolog

In situ regeneration of bioactive coatings enabled by an evolved Staphylococcus aureus sortase A

Surface immobilization of bioactive molecules is a central paradigm in the design of implantable devices and biosensors with improved clinical performance capabilities. However, in vivo degradation or denaturation of surface constituents often limits the long-term performance of bioactive films. Here we demonstrate the capacity to repeatedly regenerate a covalently immobilized monomolecular thin film of bioactive molecules through a two-step stripping and recharging cycle. Reversible transpeptidation by a laboratory evolved Staphylococcus aureus sortase A (eSrtA) enabled the rapid immobilization of an anti-thrombogenic film in the presence of whole blood and permitted multiple cycles of film regeneration in vitro that preserved its biological activity. Moreover, eSrtA transpeptidation facilitated surface re-engineering of medical devices in situ after in vivo implantation through removal and restoration film constituents. These studies establish a rapid, orthogonal and reversible biochemical scheme to regenerate selective molecular constituents with the potential to extend the lifetime of bioactive films

Solvent accessible surface area approximations for rapid and accurate protein structure prediction

The burial of hydrophobic amino acids in the protein core is a driving force in protein folding. The extent to which an amino acid interacts with the solvent and the protein core is naturally proportional to the surface area exposed to these environments. However, an accurate calculation of the solvent-accessible surface area (SASA), a geometric measure of this exposure, is numerically demanding as it is not pair-wise decomposable. Furthermore, it depends on a full-atom representation of the molecule. This manuscript introduces a series of four SASA approximations of increasing computational complexity and accuracy as well as knowledge-based environment free energy potentials based on these SASA approximations. Their ability to distinguish correctly from incorrectly folded protein models is assessed to balance speed and accuracy for protein structure prediction. We find the newly developed “Neighbor Vector” algorithm provides the most optimal balance of accurate yet rapid exposure measures

Observations of Nocturnal Foraging in the Double-crested Cormorant

Double-crested Cormorants (Phalacrocorax auritus) are normally considered a diurnal species. Here we describe cormorants foraging nocturnally in an oxbow lake in Mississippi. We have observed this behavior only once during 30 capture attempts over seven years

Double-crested Cormorant Movements in Relation to Aquaculture in Eastern Mississippi and Western Alabama

Concomitant with increasing numbers of the Double-crested Cormorant ( Phalacrocorax auritus ), catfish producers in eastern Mississippi and western Alabama have reported damage caused by cormorant predation. VHF telemetry was used to document movements of 25 cormorants from all known night roosts in the aquaculture producing areas of eastern Mississippi and western Alabama, January-April 1998. A total of 193 day locations and 396 night roost locations of the cormorants were obtained. Each cormorant was found in the study area for 57 ± 4 (SE) days. Each cormorant averaged three night roosts (range: 1-8) and spent 20 (±2) days at each night roost site. Over 95% of cormorant day locations were within 19 km of their night roosts. Catfish pond use by cormorants varied between roost sites. Cormorants from five of eleven night roosts had ≥ 30% of subsequent daytime locations on catfish ponds and birds from five of the six remaining night roosts did not visit catfish ponds on the following day. Foraging distance and frequency of night roost interchange was less for birds in this study than those reported from other aquaculture regions. We suggest roost harassment efforts should be focused on specific roost sites and some roost sites should serve as unharrassed refugia from which cormorants are less likely to cause damage to aquaculture

Reprogramming the specificity of sortase enzymes

Staphylococcusaureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore –protein–PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.Chemistry and Chemical Biolog