Side-chain thioamides as fluorescence quenching probes.

Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing Nε -thioacetyl-lysine and γ-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of Nε -thioacetyl-lysine into proteins expressed in Escherichia coli using amber codon suppression.

Chemical synthesis of Torenia plant pollen tube attractant proteins by KAHA ligation.

The synthesis of secreted cysteine-rich proteins (CRPs) is a long-standing challenge due to protein aggregation and premature formation of inter- and intramolecular disulfide bonds. Chemical synthesis provides reduced CRPs with a higher purity, which is advantageous for folding and isolation. Herein, we report the chemical synthesis of pollen tube attractant CRPs Torenia fournieri LURE (TfLURE) and Torenia concolor LURE (TcLURE) and their chimeric analogues via α-ketoacid-hydroxylamine (KAHA) ligation. The bioactivity of chemically synthesized TfLURE protein was shown to be comparable to E. coli expressed recombinant protein through in vitro assay. The convergent protein synthesis approach is beneficial for preparing these small protein variants efficiently.

Incorporating thioamides into proteins by native chemical ligation.

The thioamide is a versatile replacement of the peptide backbone with altered hydrogen bonding and conformational preferences, as well the ability participate in energy and electron transfer processes. Semi-synthetic incorporation of a thioamide into a protein can be used to study protein folding or protein/protein interactions using these properties. Semi-synthesis also provides the opportunity to study the role of thioamides in natural proteins. Here we outline the semi-synthesis of a model protein, the B1 domain of protein G (GB1) with a thioamide at the N-terminus or the C-terminus. The thioamide is synthetically incorporated into a fragment by solid-phase peptide synthesis, whereas the remainder of the protein is recombinantly expressed. Then, the two fragments are joined by native chemical ligation. The explicit protocol for GB1 synthesis is accompanied by examples of applications with GB1 and other proteins in structural biology and protein misfolding studies.

Genetic encoding of a highly photostable, long lifetime fluorescent amino acid for imaging in mammalian cells.

Acridonylalanine (Acd) is a fluorescent amino acid that is highly photostable, with a high quantum yield and long fluorescence lifetime in water. These properties make it superior to existing genetically encodable fluorescent amino acids for monitoring protein interactions and conformational changes through fluorescence polarization or lifetime experiments, including fluorescence lifetime imaging microscopy (FLIM). Here, we report the genetic incorporation of Acd using engineered pyrrolysine tRNA synthetase (RS) mutants that allow for efficient Acd incorporation in both E. coli and mammalian cells. We compare protein yields and amino acid specificity for these Acd RSs to identify an optimal construct. We also demonstrate the use of Acd in FLIM, where its long lifetime provides strong contrast compared to endogenous fluorophores and engineered fluorescent proteins, which have lifetimes less than 5 ns.