Improving target amino acid selectivity in a permissive aminoacyl tRNA synthetase through counter-selection.

The amino acid acridon-2-ylalanine (Acd) can be a valuable probe of protein dynamics, either alone or as part of a Förster resonance energy transfer (FRET) or photo-induced electron transfer (eT) probe pair. We have previously reported the genetic incorporation of Acd by an aminoacyl tRNA synthetase (RS). However, this RS, developed from a library of permissive RSs, also incorporates N-phenyl-aminophenylalanine (Npf), a trace byproduct of one Acd synthetic route. We have performed negative selections in the presence of Npf and analyzed the selectivity of the resulting AcdRSs by in vivo protein expression and detailed kinetic analyses of the purified RSs. We find that selection conferred a ∼50-fold increase in selectivity for Acd over Npf, eliminating incorporation of Npf contaminants, and allowing one to use a high yielding Acd synthetic route for improved overall expression of Acd-containing proteins. More generally, our report also provides a cautionary tale on the use of permissive RSs, as well as a strategy for improving selectivity for the target amino acid.

Characterization of the lipid binding properties of Otoferlin reveals specific interactions between PI(4,5)P2 and the C2C and C2F domains.

Otoferlin is a transmembrane protein consisting of six C2 domains, proposed to act as a calcium sensor for exocytosis. Although otoferlin is believed to bind calcium and lipids, the lipid specificity and identity of the calcium binding domains are controversial. Further, it is currently unclear whether the calcium binding affinity of otoferlin quantitatively matches the maximal intracellular presynaptic calcium concentrations of ∼30-50 µM known to elicit exocytosis. To characterize the calcium and lipid binding properties of otoferlin, we used isothermal titration calorimetry (ITC), liposome sedimentation assays, and fluorescence spectroscopy. Analysis of ITC data indicates that with the exception of the C2A domain, the C2 domains of otoferlin bind multiple calcium ions with moderate (Kd = 25-95 µM) and low affinities (Kd = 400-700 µM) in solution. However, in the presence of liposomes, the calcium sensitivity of the domains increased by up to 10-fold. It was also determined that calcium enhanced liposome binding for domains C2B-C2E, whereas the C2F domain bound liposomes in a calcium-independent manner. Mutations that abrogate calcium binding in C2F do not disrupt liposome binding, supporting the conclusion that the interaction of the C2F domain with phosphatidylserine is calcium-independent. Further, domains C2C and C2F, not domains C2A, C2B, C2D, and C2E, bound phosphatidylinositol 4,5-bisphosphate 1,2-dioleoyl-sn-glycero-3-phospho(1'-myoinositol-4',5'-bisphosphate) [PI(4,5)P2], which preferentially steered them toward liposomes harboring PI(4,5)P2. Remarkably, lysine mutations L478A and L480A in C2C selectively weaken the PI(4,5)P2 interaction while leaving phosphatidylserine binding unaffected. Finally, shifts in the emission spectra of an environmentally sensitive fluorescent unnatural amino acid indicate that the calcium binding loops of the C2F domain directly interact with the lipid bilayer of negatively charged liposomes in a calcium-independent manner. On the basis of these results, we propose that the C2F and C2C domains of otoferlin preferentially bind PI(4,5)P2 and that PI(4,5)P2 may serve to target otoferlin to the presynapse in a calcium-independent manner. This positioning would facilitate fast calcium-dependent exocytosis at the hair cell synapse.

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.

Minimalist probes for studying protein dynamics: thioamide quenching of selectively excitable fluorescent amino acids.

Fluorescent probe pairs that can be selectively excited in the presence of Trp and Tyr are of great utility in studying conformational changes in proteins. However, the size of these probe pairs can restrict their incorporation to small portions of a protein sequence where their effects on secondary and tertiary structure can be tolerated. Our findings show that a thioamide bond-a single atom substitution of the peptide backbone-can quench fluorophores that are red-shifted from intrinsic protein fluorescence, such as acridone. Using steady-state and fluorescence lifetime measurements, we further demonstrate that this quenching occurs through a dynamic electron-transfer mechanism. In a proof-of-principle experiment, we apply this technique to monitor unfolding in a model peptide system, the villin headpiece HP35 fragment. Thioamide analogues of the natural amino acids can be placed in a variety of locations in a protein sequence, allowing one to make a large number of measurements to model protein folding.

Synthesis of 2,6-disubstituted dihydropyrans via an efficient BiBr(3)-initiated three component, one-pot cascade.

The rapid synthesis of cis-2,6-disubstituted dihydropyrans is achieved in a three-component, one-pot cascade reaction. BiBr(3)-ediated addition of ketene silyl acetals or silyl enol ethers to beta,gamma-unsaturated cis-4-trimethylsilyl-3-butenal provides a Mukaiyama aldol adduct containing a vinylsilane moiety tethered to a silyl ether. Addition of a second aldehyde initiates a domino sequence involving intermolecular addition followed by an intramolecular silyl-modified Sakurai (ISMS) reaction. Isolated yields of this one-pot reaction vary from 44 to 80% and all compounds were isolated as the cis-diastereomers (10 examples).