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.

Extensive Structure-Activity Relationship Study of Albicidin's C-Terminal Dipeptidic p-Aminobenzoic Acid Moiety.

Albicidin is a recently described natural product that strongly inhibits bacterial DNA gyrase. The pronounced activity, particularly against Gram-negative bacteria, turns it into a promising lead structure for an antibacterial drug. Hence, structure-activity relationship studies are key for the in-depth understanding of structural features/moieties affecting gyrase inhibition, antibacterial activity and overcoming resistance. The 27 newly synthesized albicidins give profound insights into possibilities for variations of the C-terminus. Furthermore, in the present study, a novel derivative has been identified as overcoming resistance posed by the Klebsiella-protease AlbD. Structural modifications include, for example, azahistidine replacing the previous instable cyanoalanine as the central amino acid, as well as a triazole amide bond isostere between building blocks D and E.

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.

Label-Free On-Chip Selective Extraction of Cell-Aggregate-Laden Microcapsules from Oil into Aqueous Solution with Optical Sensor and Dielectrophoresis.

Microfluidic encapsulation of cells or tissues in biocompatible solidlike hydrogels has wide biomedical applications. However, the microfluidically encapsulated cells/tissues are usually suspended in oil and need to be extracted into aqueous solution for further culture or use. Current extracting techniques are either nonselective for the cell/tissue-laden hydrogel microcapsules or rely on fluorescence labeling of the cells/tissues, which may be undesired for their further culture or use. Here we developed a microelectromechanical system (MEMS) to achieve label-free on-chip selective extraction of cell-aggregate-laden hydrogel microcapsules from oil into aqueous solution. The system includes a microfluidic device, an optical sensor, a dielectrophoretic (DEP) actuator, and microcontrollers. The microfluidic device is for encapsulating cell aggregates in hydrogel microcapsules using the flow-focusing function with microchannels for extracting microcapsules. The optical sensor is to detect the cell aggregates, based on the difference of the optical properties between the cell aggregates and surrounding solution before their encapsulation in hydrogel microcapsules. This strategy is used because the difference in optical property between the cell-aggregate-laden hydrogel microcapsules and empty microcapsules is too small to tell them apart with a commonly used optical sensor. The DEP actuator, which is controlled by the sensor and microcontrollers, is for selectively extracting the targeted hydrogel microcapsules by DEP force. The results indicate this system can achieve selective extraction of cell-aggregate-laden hydrogel microcapsules with ∼100% efficiency without compromising the cell viability, and can improve the purity of the cell-aggregate-laden microcapsules by more than 75 times compared with nonselective extraction.

Photoactivatable Mussel-Based Underwater Adhesive Proteins by an Expanded Genetic Code.

Marine mussels exhibit potent underwater adhesion abilities under hostile conditions by employing 3,4-dihydroxyphenylalanine (DOPA)-rich mussel adhesive proteins (MAPs). However, their recombinant production is a major biotechnological challenge. Herein, a novel strategy based on genetic code expansion has been developed by engineering efficient aminoacyl-transfer RNA synthetases (aaRSs) for the photocaged noncanonical amino acid ortho-nitrobenzyl DOPA (ONB-DOPA). The engineered ONB-DOPARS enables in vivo production of MAP type 5 site-specifically equipped with multiple instances of ONB-DOPA to yield photocaged, spatiotemporally controlled underwater adhesives. Upon exposure to UV light, these proteins feature elevated wet adhesion properties. This concept offers new perspectives for the production of recombinant bioadhesives.

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.

Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coli Strain Produces a New-to-nature Bioactive Lantibiotic Containing Thienopyrrole-Alanine.

Genetic code engineering that enables reassignment of genetic codons to non-canonical amino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesized peptides and proteins with functions not commonly found in Nature. Here we report the expression of a ribosomally synthesized and post-translationally modified peptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan (Trp) residue replaced by the ncAA L-ß-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) which does not sustain cell growth in the culture. We have demonstrated that cellular toxicity of [3,2]Tpa for the production of the new-to-nature bioactive congener of lichenicidin in the host Escherichia coli can be alleviated by using an evolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but also uses it as a proteome-wide synthetic building block. This work underscores the feasibility of the biocontainment concept and establishes a general framework for design and large scale production of RiPPs with evolutionarily adapted host strains.

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.

Chemical Evolution of a Bacterial Proteome.

We have changed the amino acid set of the genetic code of Escherichia coli by evolving cultures capable of growing on the synthetic noncanonical amino acid L-ß-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) as a sole surrogate for the canonical amino acid L-tryptophan (Trp). A long-term cultivation experiment in defined synthetic media resulted in the evolution of cells capable of surviving Trp→[3,2]Tpa substitutions in their proteomes in response to the 20,899 TGG codons of the E. coli W3110 genome. These evolved bacteria with new-to-nature amino acid composition showed robust growth in the complete absence of Trp. Our experimental results illustrate an approach for the evolution of synthetic cells with alternative biochemical building blocks.

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.

Application of PBPK model for 2,4-D to estimates of risk in backpack applicators.

A PBPK model for 2,4-D was developed that involves flow-limited pH trapping modified to consider tissue binding, binding to plasma, and high-dose inhibition of urinary excretion. The PBPK model provides reasonable estimates of the kinetics of 2,4-D in rats as well as in humans, providing a common metric for expressing risk. The risk characterization for 2,4-D based on the PBPK model is consistent with that based on standard risk assessment methods, except that the apparent variability in the risk characterization is reduced. The model demonstrates that non-linear pharmacokinetics and inhibition of urinary excretion would not be expected in occupational exposures. This case study suggests that preliminary PBPK models could be developed for numerous pesticides based on commonly available data. If properly validated with well-designed worker exposure studies, such models may be useful in more complete assessments of risks to workers as well as members of the general public.