Rebecca F. Wissner

Native chemical ligation of thioamide-containing peptides: development and application to the synthesis of labeled α-synuclein for misfolding studies.

Thioamide modifications of the peptide backbone are used to perturb secondary structure, to inhibit proteolysis, as photoswitches, and as spectroscopic labels. Thus far, their incorporation has been confined to single peptides synthesized on solid phase. We have generated thioamides in C-terminal thioesters or N-terminal Cys fragments and examined their compatibility with native chemical ligation conditions. Most sequence variants can be coupled in good yields with either TCEP or DTT as the reductant, though some byproducts are observed with prolonged TCEP incubations. Furthermore, we find that thioamides are compatible with thiazolidine protection of an N-terminal Cys, so that multiple ligations can be used to construct larger proteins. Since the acid-lability of the thioamide prohibits on-resin thioester synthesis using Boc chemistry, we devised a method for the synthesis of thioamide peptides with a masked C-terminal thioester that is revealed in situ. Finally, we have shown that thioamidous peptides can be coupled to expressed protein fragments to generate large proteins with backbone thioamide labels by synthesizing labeled versions of the amyloid protein α-synuclein for protein folding studies. In a proof-of-principle experiment, we demonstrated that quenching of fluorescence by thioamides can be used to track conformational changes during aggregation of labeled α-synuclein.

Labeling proteins with fluorophore/thioamide Förster resonant energy transfer pairs by combining unnatural amino acid mutagenesis and native chemical ligation.

We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Förster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.

Efficient Synthesis and In Vivo Incorporation of Acridon-2-ylalanine, a Fluorescent Amino Acid for Lifetime and Förster Resonance Energy Transfer/Luminescence Resonance Energy Transfer Studies

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein conformational change because it is a long lifetime, visible wavelength fluorophore that is small enough to be incorporated during ribosomal biosynthesis. Incorporation of Acd into proteins expressed in Escherichia coli requires efficient chemical synthesis to produce large quantities of the amino acid and the generation of a mutant aminoacyl tRNA synthetase that can selectively charge the amino acid onto a tRNA. Here, we report the synthesis of Acd in 87% yield over five steps from Tyr and the identification of an Acd synthetase by screening candidate enzymes previously evolved from Methanococcus janaschii Tyr synthetase for unnatural amino acid incorporation. Furthermore, we characterize the photophysical properties of Acd, including quenching interactions with select natural amino acids and Förster resonance energy transfer (FRET) interactions with common fluorophores such as methoxycoumarin (Mcm). Finally, we demonstrate the value of incorporation of Acd into proteins, using changes in Acd fluorescence lifetimes, Mcm/Acd FRET, or energy transfer to Eu(3+) to monitor protein folding and binding interactions.

Multiply labeling proteins for studies of folding and stability

Fluorescence spectroscopy is a powerful method for monitoring protein folding in real-time with high resolution and sensitivity, but requires the site-specific introduction of labels into the protein. The ability to genetically incorporate unnatural amino acids (Uaas) allows for the efficient synthesis of fluorescently labeled proteins with minimally perturbing fluorophores. Here, we describe recent uses of labeled proteins in dynamic structure determination experiments and advances in unnatural amino acid incorporation for dual site-specific fluorescent labeling. The advent of increasingly sophisticated bioorthogonal chemistry reactions and the diversity of Uaas available for incorporation will greatly enable protein folding and stability studies.

Site-Specific Fluorescence Polarization for Studying the Disaggregation of α-Synuclein Fibrils by Small Molecules

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinsons disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinsons disease.

Investigating the Trimethylamine N-Oxide (TMAO) Induced Structure of α-Synuclein

Trimethylamine N-oxide (TMAO) is a naturally occurring osmolyte that is known to stabilize protein structure. Previous studies have shown that the addition of TMAO can induce folding of thermodynamically unstable proteins, causing them to regain high functional activity. In solution, monomeric α-synuclein (αS) is intrinsically disordered. Our laboratory and others have shown that αS undergoes significant compaction in the presence of TMAO. Previously, we have demonstrated that p-cyanophenylalanine and a thioamide can serve as a minimally perturbing probe pair for Forster Resonance Energy Transfer (FRET) experiments. Despite the utility of this pair in measuring short intramolecular distances, inclusion of the thioamide is synthetically intensive, rendering it difficult to generate a large library of double-labeled mutants for FRET studies. As an alternative, we have expressed a library of double-labeled αS mutants containing the genetically encodable FRET pair, Cnf and tryptophan (Trp). This set of double-labeled proteins will allow us to obtain a more comprehensive description of the TMAO-induced morphology of αS.