Through bonds or contacts? Mapping protein vibrational energy transfer using non-canonical amino acids.

Vibrational energy transfer (VET) is essential for protein function. It is responsible for efficient energy dissipation in reaction sites, and has been linked to pathways of allosteric communication. While it is understood that VET occurs via backbone as well as via non-covalent contacts, little is known about the competition of these two transport channels, which determines the VET pathways. To tackle this problem, we equipped the ß-hairpin fold of a tryptophan zipper with pairs of non-canonical amino acids, one serving as a VET injector and one as a VET sensor in a femtosecond pump probe experiment. Accompanying extensive non-equilibrium molecular dynamics simulations combined with a master equation analysis unravel the VET pathways. Our joint experimental/computational endeavor reveals the efficiency of backbone vs. contact transport, showing that even if cutting short backbone stretches of only 3 to 4 amino acids in a protein, hydrogen bonds are the dominant VET pathway.

Site-Resolved Observation of Vibrational Energy Transfer Using a Genetically Encoded Ultrafast Heater.

Allosteric information transfer in proteins has been linked to distinct vibrational energy transfer (VET) pathways in a number of theoretical studies. Experimental evidence for such pathways, however, is sparse because site-selective injection of vibrational energy into a protein, that is, localized heating, is required for their investigation. Here, we solved this problem by the site-specific incorporation of the non-canonical amino acid ß-(1-azulenyl)-l-alanine (AzAla) through genetic code expansion. As an exception to Kasha's rule, AzAla undergoes ultrafast internal conversion and heating after S1 excitation while upon S2 excitation, it serves as a fluorescent label. We equipped PDZ3, a protein interaction domain of postsynaptic density protein 95, with this ultrafast heater at two distinct positions. We indeed observed VET from the incorporation sites in the protein to a bound peptide ligand on the picosecond timescale by ultrafast IR spectroscopy. This approach based on genetically encoded AzAla paves the way for detailed studies of VET and its role in a wide range of proteins.

Broad substrate tolerance of tubulin tyrosine ligase enables one-step site-specific enzymatic protein labeling.

The broad substrate tolerance of tubulin tyrosine ligase is the basic rationale behind its wide applicability for chemoenzymatic protein functionalization. In this context, we report that the wild-type enzyme enables ligation of various unnatural amino acids that are substantially bigger than and structurally unrelated to the natural substrate, tyrosine, without the need for extensive protein engineering. This unusual substrate flexibility is due to the fact that the enzyme's catalytic pocket forms an extended cavity during ligation, as confirmed by docking experiments and all-atom molecular dynamics simulations. This feature enabled one-step C-terminal biotinylation and fluorescent coumarin labeling of various functional proteins as demonstrated with ubiquitin, an antigen binding nanobody, and the apoptosis marker Annexin V. Its broad substrate tolerance establishes tubulin tyrosine ligase as a powerful tool for in vitro enzyme-mediated protein modification with single functional amino acids in a specific structural context.

Toward intrinsically colored peptides: Synthesis and investigation of the spectral properties of methylated azatryptophans in tryptophan-cage mutants.

Tryptophan has been taken as the basic scaffold for a chromophore whose indole residue can be further functionalized by the introduction of endocyclic nitrogen atoms or by N-methylation. When compared with exocyclic modifications, modifying tryptophan in an endocyclic fashion (through atomic substitution) should not perturb the steric profile of the amino acid side chain to such a large extent as that of an exocyclic modification, while simultaneously modulating the polarity, hydrogen-bonding ability, and spectral properties of the amino acid. Of particular interest is that the spectral properties can be tailored such that the chromophore can be monitored at wavelengths that exceed natural protein fluorescence. Ideally, the optimum excitation wavelength should be between 300 and 350 nm, and the emission wavelength should be ≥500 nm such that no cross-excitation/fluorescence occurs. Here, we report the synthesis of amino acid labels that exhibit large red shifts in their fluorescence profiles and their use in peptides.

Intramolecular 1,6-addition to 2-pyridones. Mechanism and synthetic scope.

The scope and limitations of the intramolecular 1,6-addition of an enolate to a 2-pyridone moiety, a reaction that has found application in the synthesis of the lupin alkaloids, have been probed. This nucleophilic addition process has been shown to be reversible and favored in the case of (less stabilized) amide and lactam enolates, which readily form five- and six-membered bi-/tricyclic products. Alternative enolates (ketone, ester, thiolactam) and a variety of different acceptors (isoquinolinone, pyrimidinone, pyrazinone, pyridopyrazinone) have been evaluated, and a range of competing side reactions have been identified and characterized using various techniques, including in situ IR.