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The aromatic side-chains of phenylalanine, tyrosine, and tryptophan interact with their environments via both hydrophobic and electrostatic interactions. Determining the extent to which these contribute to protein function and stability is not possible with conventional mutagenesis. Serial fluorination of a given aromatic is a validated method in vitro and in silico to specifically alter electrostatic characteristics, but this approach is restricted to a select few experimental systems. Here, we report a group of pyrrolysine-based aminoacyl-tRNA synthetase/tRNA pairs (tRNA/RS pairs) that enable the site-specific encoding of a varied spectrum of fluorinated phenylalanine amino acids in E. coli and mammalian (HEK 293T) cells. By allowing the cross-kingdom expression of proteins bearing these unnatural amino acids at biochemical scale, these tools may potentially enable the study of biological mechanisms which utilize aromatic interactions in structural and cellular contexts. Aromatic amino acids in proteins support ligand binding and protein stability. To parse the physiocochemical roles of aromatic interactions, here Galles, Infield and co-authors identify pyrrolysine-based aminoacyl-tRNA synthetases that enable the encoding of fluorinated phenylalanine amino acids.

With the recent explosion in high-resolution protein structures, one of the next frontiers in biology is elucidating the mechanisms by which conformational rearrangements in proteins are regulated to meet the needs of cells under changing conditions. Rigorously measuring protein energetics and dynamics requires the development of new methods that can resolve structural heterogeneity and conformational distributions. We have previously developed steady-state transition metal ion fluorescence resonance energy transfer (tmFRET) approaches using a fluorescent noncanonical amino acid donor (Anap) and transition metal ion acceptor to probe conformational rearrangements in soluble and membrane proteins. Here, we show that the fluorescent noncanonical amino acid Acd has superior photophysical properties that extend its utility as a donor for tmFRET. Using maltose-binding protein (MBP) expressed in mammalian cells as a model system, we show that Acd is comparable to Anap in steady-state tmFRET experiments and that its long, single-exponential lifetime is better suited for probing conformational distributions using time-resolved FRET. These experiments reveal differences in heterogeneity in the apo and holo conformational states of MBP and produce accurate quantification of the distributions among apo and holo conformational states at subsaturating maltose concentrations. Our new approach using Acd for time-resolved tmFRET sets the stage for measuring the energetics of conformational rearrangements in soluble and membrane proteins in near-native conditions.

Ligands such as insulin, epidermal growth factor, platelet-derived growth factor, and nerve growth factor (NGF) initiate signals at the cell membrane by binding to receptor tyrosine kinases (RTKs). Along with G-protein-coupled receptors, RTKs are the main platforms for transducing extracellular signals into intracellular signals. Studying RTK signaling has been a challenge, however, due to the multiple signaling pathways to which RTKs typically are coupled, including MAP/ERK, PLCγ, and Class 1A phosphoinositide 3-kinases (PI3K). The multi-pronged RTK signaling has been a barrier to isolating the effects of any one downstream pathway. Here, we used optogenetic activation of PI3K to decouple its activation from other RTK signaling pathways. In this context, we used genetic code expansion to introduce a click chemistry noncanonical amino acid into the extracellular side of membrane proteins. Applying a cell-impermeant click chemistry fluorophore allowed us to visualize delivery of membrane proteins to the plasma membrane in real time. Using these approaches, we demonstrate that activation of PI3K, without activating other pathways downstream of RTK signaling, is sufficient to traffic the TRPV1 ion channels and insulin receptors to the plasma membrane.

Eurythmy Therapy (ET) is a mindfulness oriented therapy developed in the context of anthroposophic medicine. Despite commonly used in practice, it remains unclear whether active participation (Inner Correspondence) during ET can be observed in eurythmy gestures (EGest). So far, no validated peer-report instrument to evaluate EGest exists. To validate an 83-item ET peer-report scale, a nested study on a sample of n = 82 breast cancer survivors with cancer-related fatigue was conducted. EGest were evaluated twice, at baseline and at 10-week follow-up, by peer-reports from two separate therapists. Interrater-reliability (IRR) was estimated by Cohen’s weighted kappa (κw) across all items. Additionally, reliability-(RA) and principal component analyses (PCA) were conducted. Patients completed two self-report scales: Satisfaction with ET (SET) and Inner Correspondence with the Movement Therapy (ICPH). IRR was greater than or equal (κw ≥ 0.25) for 41 items (49.3%) with a mean weighted kappa of κ̅w = 0.40 (SD = 0.17, range = 0.25–0.85). RA resulted in the exclusion of 25 items with insufficient item-total correlations < 0.40. A PCA with 16 items revealed 3 subscales: 1. Mindfulness in Movement (8 items), 2. Motor Skills (5 items), 3. Walking Pattern (3items) explaining 63.86% of total variance. Internal consistency (Cronbach’s alpha) was high for the sum score with α = 0.89 and for the subscales with α = 0.88, 0.86 and 0.84 respectively. Significant small to moderate subscale correlations were found ranging from r = 0.29–0.63 (all p < 0.01). Mindfulness in Movement correlated with Inner Correspondence (r = 0.32) and with Satisfaction with ET (r = - 0.25, both p < 0.05). The new AART-ASSESS-EuMove is the first consistent and reliable peer-report instrument to evaluate EGest. It shows associations between peer-reported Mindful Movement and patients’ self-reported ICPH and SET. •Eurythmy therapy (ET) is an anthroposophic mindfulness-oriented therapy.•The peer-review instrument: Anthroposophic Artistic Movement Assessment for Eurythmy Therapy (AART-ASSESS-EuMove) was validated.•Items with sufficient interrater reliability formed the subscales: Mindfulness in Movement, Motor Skills and Walking Pattern.•The AART-ASSESS-EuMove showed sufficient internal consistency and correlates with patient-reported outcome.•The new instrument broadens methodological options of questionnaire validation and contributes to the understanding of ET.

Stable surface adhesion of cells is one of the early pivotal steps in bacterial biofilm formation, a prevalent adaptation strategy in response to changing environments. In Pseudomonas fluorescens, this process is regulated by the Lap system and the second messenger cyclic-di-GMP. High cytoplasmic levels of cyclic-di-GMP activate the transmembrane receptor LapD that in turn recruits the periplasmic protease LapG, preventing it from cleaving a cell surface-bound adhesin, thereby promoting cell adhesion. In this study, we elucidate the molecular basis of LapG regulation by LapD and reveal a remarkably sensitive switching mechanism that is controlled by LapD's HAMP domain. LapD appears to act as a coincidence detector, whereby a weak interaction of LapG with LapD transmits a transient outside-in signal that is reinforced only when cyclic-di-GMP levels increase. Given the conservation of key elements of this receptor system in many bacterial species, the results are broadly relevant for cyclic-di-GMP- and HAMP domain-regulated transmembrane signaling. While bacteria often live as unicellular microorganisms, many bacteria are capable of sticking together on a surface and forming a multicellular structure called a biofilm. Bacterial biofilms occur frequently in nature; for example, on the roots of plants and submerged rocks. While these biofilms are generally innocuous, others pose significant health threats to humans, causing tooth decay, gum disease, and—when they occur on implanted devices such as prosthetic heart valves—potentially serious infections. When in biofilms, many bacteria are tolerant to antibiotics; therefore, working out how to disrupt these films is crucial for developing new treatments. The microorganism Pseudomonas fluorescens is an example of a bacterium that can be found living in a complex biofilm. In response to certain environmental cues, free-swimming P. fluorescens cells adhere to a surface and produce a slime that encases them in a robust biofilm. The decision to shift between a free-swimming and a biofilm life-style is orchestrated by a signaling molecule found inside the bacteria called cyclic-di-GMP. In P. fluorescens, the availability of nutrients—in particular, phosphate—controls how much cyclic-di-GMP is produced inside the cell. If not enough phosphate is available, the level of cyclic-di-GMP falls and the biofilm disperses. Cyclic-di-GMP affects the stability of the biofilm via a group of proteins called the Lap system. When levels of cyclic-di-GMP are high, cyclic-di-GMP binds to a protein called LapD, which can then in turn bind to an enzyme known as LapG. When bound to LapD, LapG is unable to break apart the molecules that help P. fluorescens cells bind to a surface, and so a biofilm can form. If cyclic-di-GMP levels drop, fewer LapD molecules can bind to cyclic-di-GMP. As cyclic-di-GMP-unbound LapD proteins interact poorly with LapG, this leaves some LapG molecules able to destabilize the attachments between the cells and the surface, which disperses the biofilm. Here, Chatterjee et al. reveal the molecular mechanism by which LapD and LapG interact in P. fluorescens. When cyclic-di-GMP is bound to LapD, the shape of LapD changes to produce features that fit into the surface of LapG. It is this shape compatibility, more so than an increase in the number or quality of interactions between the chemical groups that make up the proteins, that enables LapD to bind to LapG. Chatterjee et al. also provide evidence that the LapD–LapG interaction can be disrupted, thereby raising the possibility that biofilm formation could be manipulated by targeting this system. Given that systems similar to the P. fluorescens Lap system exist in numerous other bacterial species, including important pathogens, the findings of Chatterjee et al. could assist efforts to develop medicines and products that eradicate bacterial biofilms. LapD also shares many structural elements with a large number of other signaling proteins; therefore, these findings could also improve the understanding of how other cell signaling systems work.

The development of bioorthogonal fluorogenic probes constitutes a vital force to advance life sciences. Tetrazine‐encoded green fluorescent proteins (GFPs) show high bioorthogonal reaction rate and genetic encodability but suffer from low fluorogenicity. Here, we unveil the real‐time fluorescence mechanisms by investigating two site‐specific tetrazine‐modified superfolder GFPs via ultrafast spectroscopy and theoretical calculations. Förster resonance energy transfer is quantitatively modeled and revealed to govern the fluorescence quenching; for GFP150‐Tet with a fluorescence turn‐on ratio of ∼9, it contains trimodal subpopulations with good (P1), random (P2), and poor (P3) alignments between the transition dipole moments of protein chromophore (donor) and tetrazine tag (Tet‐v2.0, acceptor). By rationally designing a more free/tight environment, we created new mutants Y200A/S202Y to introduce more P2/P1 populations and improve the turn‐on ratios to ∼14/31, making the fluorogenicity of GFP150‐Tet‐S202Y the highest among all up‐to‐date tetrazine‐encoded GFPs. In live eukaryotic cells, the GFP150‐Tet‐v3.0‐S202Y mutant demonstrates notably increased fluorogenicity, substantiating our generalizable design strategy. Key points Ultrafast spectroscopy reveals FRET in action and inhomogeneous populations with different transition dipole moment alignments. Rational protein design of two new superfolder GFP mutants with record‐high fluorogenicity. Bioimaging application of the designed bioorthogonal protein mutant in live eukaryotic cells. Targeted mutagenesis aided by ultrafast molecular spectroscopic insights into the tetrazine‐encoded green fluorescent proteins in action in physiologically relevant environments has effectively achieved the record‐high bioorthogonal fluorogenicity to date for next‐generation bioimaging and life science advances. The rational protein design strategy incorporating noncanonical amino acids with a gain of function and demonstrated live eukaryotic cell applications is generalizable to any fluorescence‐based detectors and sensors.

Wear phenomena of ceramic inlays are not fully understood. The aim of the present study was to evaluate ceramic wear, antagonist enamel wear, and luting cement wear over 8 years. The two-fold null hypothesis was that there would be (1) no difference in wear behavior between ceramic and enamel, and (2) no influence of filler content of luting composites on composite wear. From 96 restorations, 36 Class II inlays from 16 participants were selected. For inlays with opposing enamel cusps (n = 17), replicas of inlays and enamel were scanned with a 3-D laser scanner. Luting gaps of inlays (n = 36) were analyzed with a profilometer, including 3-D data analysis. Ceramic and enamel wear increased between 4 and 8 years, with significantly higher values for enamel after 6 years (p < 0.05). Luting gap wear increased continuously up to 8 years (p < 0.05), with no influence of luting composites (p > 0.05) and location of teeth (p > 0.05).

The Arroyo La Estacada (~ 33°28' S, 69°02' W), eastern Andean piedmont of Argentina, cuts through an extensive piedmont aggradational unit composed of a dominant Late Pleistocene–early Holocene (LP–EH) alluvial sequence that includes several paleosols. One of these paleosols developed affecting the topmost part of likely Late Glacial aeolian deposits aggraded into a floodplain environment by the end of the Late Pleistocene. The paleosol shows variable grade of development along the arroyo outcrops. Organic matter humification, carbonate accumulation and redox processes were the dominant processes associated with paleosol formation. By the early Holocene, when the formation of the paleosol ended, renewed alluvial aggradation and high magnitude flooding events affected the arroyo's floodplain environment. Accordignly, a period of relative landscape stability in the Arroyo La Estacada basin is inferred from the paleosol developed by the LP–EH transition in response to the climatic conditions in the Andes cordillera piedmont after the Late Glacial arid conditions. The analyzed Late Glacial–Holocene alluvial record of the Andean piedmont constitutes a suitable record of the LP–EH climatic transition in the extra-Andean region of Argentina. It is in agreement with regional paleoclimatic evidence along the southern tip of the South American continent, where other pedosedimentary sequences record similar late Quaternary paleoenvironmental changes over both fluvial and interfluvial areas.

The ability to introduce different biophysical probes into defined positions in target proteins will provide powerful approaches for interrogating protein structure, function and dynamics. However, methods for site-specifically incorporating multiple distinct unnatural amino acids are hampered by their low efficiency. Here we provide a general solution to this challenge by developing an optimized orthogonal translation system that uses amber and evolved quadruplet-decoding transfer RNAs to encode numerous pairs of distinct unnatural amino acids into a single protein expressed in Escherichia coli with a substantial increase in efficiency over previous methods. We also provide a general strategy for labelling pairs of encoded unnatural amino acids with different probes via rapid and spontaneous reactions under physiological conditions. We demonstrate the utility of our approach by genetically directing the labelling of several pairs of sites in calmodulin with fluorophores and probing protein structure and dynamics by Förster resonance energy transfer. A series of quadruplet decoding tRNAs has been developed to form an optimized orthogonal translation system. These tRNAs enable efficient, site-specific incorporation of multiple unnatural amino acids into a protein, with a substantial increase in yield over previous methods. The amino acids are then used to site-specifically label a protein with a pair of fluorophores, enabling studies of the protein's dynamics.

A straightforward protocol for the site-specific incorporation of a 19 F label into any protein in vivo is described. This is done using a plasmid containing an orthogonal aminoacyl-tRNA synthetase/tRNA CUA that incorporates L -4-trifluoromethylphenylalanine in response to the amber codon UAG. This method improves on other in vivo methods because the 19 F label is incorporated into only one location on the protein of interest and that protein can easily be produced in large quantities at low cost. The protocol for producing 19 F-labeled protein is similar to expressing protein in Escherichia coli and takes 4 d to obtain pure protein starting from the appropriate vectors.