Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles.

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

A framework for establishing restoration goals for contaminated ecosystems.

As natural resources become increasingly limited, the value of restoring contaminated sites, both terrestrial and aquatic, becomes increasingly apparent. Traditionally, goals for remediation have been set before any consideration of goals for ecological restoration. The goals for remediation have focused on removing or limiting contamination whereas restoration goals have targeted the ultimate end use. Here, we present a framework for developing a comprehensive set of achievable goals for ecological restoration of contaminated sites to be used in concert with determining goals for remediation. This framework was developed during a Society of Environmental Toxicology and Chemistry (SETAC) and Society of Ecological Restoration (SER) cosponsored workshop that brought together experts from multiple countries. Although most members were from North America, this framework is designed for use internationally. We discuss the integration of establishing goals for both contaminant remediation and overall restoration, and the need to include both the restoration of ecological and socio-cultural-economic value in the context of contaminated sites. Although recognizing that in some countries there may be regulatory issues associated with contaminants and clean up, landscape setting and social drivers can inform the restoration goals. We provide a decision tree support tool to guide the establishment of restoration goals for contaminated ecosystems. The overall intent of this decision tree is to provide a framework for goal setting and to identify outcomes achievable given the contamination present at a site.

Transferase-Mediated Labeling of Protein N-Termini with Click Chemistry Handles.

The E. coli aminoacyl transferase (AaT) can be used to transfer a variety of unnatural amino acids, including those with azide or alkyne groups, to the α-amine of a protein with an N-terminal Lys or Arg. Subsequent functionalization through either copper-catalyzed or strain-promoted click reactions can be used to label the protein with fluorophores or biotin. This method can be used to directly detect AaT substrates or in a two-step protocol to detect substrates of the mammalian ATE1 transferase.

Efficient, Traceless Semi-Synthesis of α-Synuclein Labeled with a Fluorophore/Thioamide FRET Pair.

We have shown that thioamides can be incorporated into proteins through semi-synthesis and used as probes to monitor structural changes. To date, our methods have required the presence of a cysteine at the peptide ligation site, which may not be present in the native peptide sequence. Here, we present a strategy for the semi-synthesis of thioproteins using homocysteine as a ligation point with subsequent masking as methionine, making the ligation "traceless."

N-terminal protein modification using simple aminoacyl transferase substrates.

Methods for synthetically manipulating protein structure enable greater flexibility in the study of protein function. Previous characterization of the Escherichia coli aminoacyl tRNA transferase (AaT) has shown that it can modify the N-terminus of a protein with an amino acid from a tRNA or a synthetic oligonucleotide donor. Here, we demonstrate that AaT can efficiently use a minimal adenosine substrate, which can be synthesized in one to two steps from readily available starting materials. We have characterized the enzymatic activity of AaT with aminoacyl adenosyl donors and found that reaction products do not inhibit AaT. The use of adenosyl donors removes the substrate limitations imposed by the use of synthetases for tRNA charging and avoids the complex synthesis of an oligonucleotide donor. Thus, our AaT donors increase the potential substrate scope and reaction scale for N-terminal protein modification under conditions that maintain folding.