Mapping the role of aromatic amino acids within a blue-light sensing LOV domain

Photosensing LOV (Light, Oxygen, Voltage) domains detect and respond to UVA/Blue (BL) light by forming a covalent adduct between the flavin chromophore and a nearby cysteine, via the decay of the flavin triplet excited state. LOV domains where the reactive cysteine has been mutated are valuable fluorescent tools for microscopy and as genetically encoded photosensitisers for reactive oxygen species. Besides being convenient tools for applications, LOV domains without the reactive cysteine (naturally occurring or engineered) can still be functionally photoactivated via formation of a neutral flavin radical. Tryptophans and tyrosines are held as the main partners as potential electron donors to the flavin excited states. In this work, we explore the relevance of aromatic amino acids in determining the photophysical features of the LOV protein Mr4511 from Methylobacterium radiotolerans by introducing point mutations into the C71S variant that does not form the covalent adduct. By using an array of spectroscopic techniques we measured the fluorescence quantum yields and lifetimes, the triplet yields and lifetimes, and the efficiency of singlet oxygen (SO) formation for eleven Mr4511 variants. Insertion of Trp residues at distances between 0.6 and 1.5 nm from the flavin chromophore results in strong quenching of the flavin excited triplet state and, at the shorter distances even of the singlet excited state. The mutation F130W (ca. 0.6 nm) completely quenches the singlet excited state, preventing triplet formation: in this case, even if the cysteine is present, the photo-adduct is not formed. Tyrosines are also quenchers for the flavin excited states, although not as efficient as Trp residues, as demonstrated with their substitution with the inert phenylalanine. For one of these variants, C71S/Y116F, we found that the quantum yield of formation for singlet oxygen is 0.44 in aqueous aerobic solution, vs 0.17 for C71S. Based on our study with Mr4511 and on literature data for other LOV domains we suggest that Trp and Tyr residues too close to the flavin chromophore (at distances less than 0.9 nm) reduce the yield of photoproduct formation and that introduction of inert Phe residues in key positions can help in developing efficient, LOV-based photosensitisers. This journal i

A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination

A T cell mounts an immune response by measuring the binding strength of its T cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T cell signaling

Formation of targeted monovalent quantum dots by steric exclusion

Precise control over interfacial chemistry between nanoparticles and other materials remains a significant challenge limiting the broad application of nanotechnology in biology. To address this challenge, we use “Steric Exclusion” to completely convert commercial quantum dots (QDs) into monovalent imaging probes by wrapping the QD with a functionalized oligonucleotide. We demonstrate the utility of these QDs as modular and non-perturbing imaging probes by tracking individual Notch receptors on live cells

Correlated Photon-Pair Emission from a Charged Single Quantum Dot

The optical creation and recombination of charged biexciton and trion complexes in an (In,Ga)As/GaAs quantum dot is investigated by micro-photoluminescence spectroscopy. Photon cross-correlation measurements demonstrate the temporally correlated decay of charged biexciton and trion states. Our calculations provide strong evidence for radiative decay from the excited trion state which allows for a deeper insight into the spin configurations and their dynamics in these systems.Comment: 5 pages, 3 figures, submitted for publicatio

Liquid-liquid transition in water from first principles

A longstanding question in water research is the possibility that supercooled liquid water can undergo a liquid-liquid phase transition (LLT) into high- and low-density liquids. We used several complementary molecular simulation techniques to evaluate the possibility of an LLT in an ab initio neural network model of water trained on density functional theory calculations with the SCAN exchange correlation functional. We conclusively show the existence of a first-order LLT and an associated critical point in the SCAN description of water, representing the first definitive computational evidence for an LLT in water from first principles.Comment: 14 pages, 3 figures. Supplemental material contains 11 pages, 8 figure

Dispersion force for materials relevant for micro and nanodevices fabrication

The dispersion (van der Waals and Casimir) force between two semi-spaces are calculated using the Lifshitz theory for different materials relevant for micro and nanodevices fabrication, namely, gold, silicon, gallium arsenide, diamond and two types of diamond-like carbon (DLC), silicon carbide, silicon nitride and silicon dioxide. The calculations were performed using recent experimental optical data available in the literature, usually ranging from the far infrared up to the extreme ultraviolet bands of the electromagnetic spectrum. The results are presented in the form of a correction factor to the Casimir force predicted between perfect conductors, for the separation between the semi-spaces varying from 1 nanometre up to 1 micrometre. The relative importance of the contributions to the dispersion force of the optical properties in different spectral ranges is analyzed. The role of the temperature for semiconductors and insulators is also addressed. The results are meant to be useful for the estimation of the impact of the Casimir and van der Waals forces on the operational parameters of micro and nanodevices

Reactivity-Dependent PCR: Direct, Solution-Phase in Vitro Selection for Bond Formation

In vitro selection is a key component of efforts to discover functional nucleic acids and small molecules from libraries of DNA, RNA, and DNA-encoded small molecules. Such selections have been widely used to evolve RNA and DNA catalysts and, more recently, to discover new reactions from DNA-encoded libraries of potential substrates. While effective, current strategies for selections of bond-forming and bond-cleaving reactivity are generally indirect, require the synthesis of biotin-linked substrates, and involve multiple solution-phase and solid-phase manipulations. In this work we report the successful development and validation of reactivity-dependent PCR (RDPCR), a new method that more directly links bond formation or bond cleavage with the amplification of desired sequences and that obviates the need for solid-phase capture, washing, and elution steps. We show that RDPCR can be used to select for bond formation in the context of reaction discovery and for bond cleavage in the context of protease activity profiling.Chemistry and Chemical Biolog

Interaction-Dependent PCR: Identification of Ligand−Target Pairs from Libraries of Ligands and Libraries of Targets in a Single Solution-Phase Experiment

Interaction-dependent PCR (IDPCR) is a solution-phase method to identify binding partners from combined libraries of small-molecule ligands and targets in a single experiment. Binding between DNA-linked targets and DNA-linked ligands induces formation of an extendable duplex. Extension links codes that identify the ligand and target into one selectively amplifiable DNA molecule. In a model selection, IDPCR resulted in the enrichment of DNA encoding all five known protein−ligand pairs out of 67 599 possible sequences.Chemistry and Chemical Biolog