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 E. 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 M. 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 Eu3+ to monitor protein folding and binding interactions.

Summary Super-resolution microscopy requires that subcellular structures are labeled with bright and photostable fluorophores, especially for live-cell imaging. Organic fluorophores may help here as they can yield more photons—by orders of magnitude—than fluorescent proteins. To achieve molecular specificity with organic fluorophores in live cells, self-labeling proteins are often used, with HaloTags and SNAP-tags being the most common. However, how these two different tagging systems compare with each other is unclear, especially for stimulated emission depletion (STED) microscopy, which is limited to a small repertoire of fluorophores in living cells. Herein, we compare the two labeling approaches in confocal and STED imaging using various proteins and two model systems. Strikingly, we find that the fluorescent signal can be up to 9-fold higher with HaloTags than with SNAP-tags when using far-red rhodamine derivatives. This result demonstrates that the labeling strategy matters and can greatly influence the duration of super-resolution imaging.

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.

New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported “cell-penetrating peptides” (CPPs) transport a model protein cargo—the self-labeling enzyme SNAP-tag—beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo—the engineered peroxidase APEX2—and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales.

Thioamides quench tryptophan and tyrosine fluorescence in a distance-dependent manner and thus can be used to monitor the binding of thioamide-containing peptides to proteins. Since thioamide analogs of the natural amino acids can be synthetically incorporated into peptides, they can function as minimally-perturbing probes of protein/peptide interactions.