Living cells are complex, crowded, and dynamic and continually respond to environmental and intracellular stimuli. They also have heterogeneous ionic strength with compartmentalized variations in both intracellular concentrations and types of ions. These challenges would benefit from the development of quantitative, noninvasive approaches for mapping the heterogeneous ionic strength fluctuations in living cells. Here, we investigated a class of recently developed ionic strength sensors that consists of mCerulean3 (a cyan fluorescent protein) and mCitrine (a yellow fluorescent protein) tethered via a linker made of two charged α-helices and a flexible loop. The two helices are designed to bear opposite charges, which is hypothesized to increase the ionic screening and therefore a larger intermolecular distance. In these protein constructs, mCerulean3 and mCitrine act as a donor–acceptor pair undergoing Förster resonance energy transfer (FRET) that is dependent on both the linker amino acids and the environmental ionic strength. Using time-resolved fluorescence of the donor (mCerulean3), we determined the sensitivity of the energy transfer efficiencies and the donor–acceptor distances of these sensors at variable concentrations of the Hofmeister series of salts (KCl, LiCl, NaCl, NaBr, NaI, Na2SO4). As controls, similar measurements were carried out on the FRET-incapable, enzymatically cleaved counterparts of these sensors as well as a construct designed with two electrostatically neutral α-helices (E6G2). Our results show that the energy transfer efficiencies of these sensors are sensitive to both the linker amino acid sequence and the environmental ionic strength, whereas the sensitivity of these sensors to the identity of the dissolved ions of the Hofmeister series of salts seems limited. We also developed a theoretical framework to explain the observed trends as a function of the ionic strength in terms of the Debye screening of the electrostatic interaction between the two charged α-helices in the linker region. These controlled solution studies represent an important step toward the development of rationally designed FRET-based environmental sensors while offering different models for calculating the energy transfer efficiency using time-resolved fluorescence that is compatible with future in vivo studies.

In this report, we have developed a simple approach using single-detector fluorescence autocorrelation spectroscopy (FCS) to investigate the Förster resonance energy transfer (FRET) of genetically encoded, freely diffusing crTC2.1 (mTurquoise2.1–linker–mCitrine) at the single molecule level. We hypothesize that the molecular brightness of the freely diffusing donor (mTurquoise2.1) in the presence of the acceptor (mCitrine) is lower than that of the donor alone due to FRET. To test this hypothesis, the fluorescence fluctuation signal and number of molecules of freely diffusing construct were measured using FCS to calculate the molecular brightness of the donor, excited at 405 nm and detected at 475/50 nm, in the presence and absence of the acceptor. Our results indicate that the molecular brightness of cleaved crTC2.1 in a buffer is larger than that of the intact counterpart under 405-nm excitation. The energy transfer efficiency at the single molecule level is larger and more spread in values as compared with the ensemble-averaging time-resolved fluorescence measurements. In contrast, the molecular brightness of the intact crTC2.1, under 488 nm excitation of the acceptor (531/40 nm detection), is the same or slightly larger than that of the cleaved counterpart. These FCS-FRET measurements on freely diffusing donor-acceptor pairs are independent of the precise time constants associated with autocorrelation curves due to the presence of potential photophysical processes. Ultimately, when used in living cells, the proposed approach would only require a low expression level of these genetically encoded constructs, helping to limit potential interference with the cell machinery.

High throughput (HTCD) using 96 and 384 multiplate for the optimisation of protein crystallization conditions, protein-ligand binding screening, and CD Imaging (CDi) of thin films of materials (proteins, nucleic acids, carbohydrates, polymers with embedded drugs, organic LED, and in general any chiral material with or without achiral substrates) are the results of the unique highly collimated microbeam light generated at B23 beamline at Diamond Light Source in the vacuum UV to visible wavelength range (130-650nm). B23 is the only bespoke beamline worldwide to characterise the structural and conformational properties of biologically important molecules using micro-devices unattainable with bespoke bench-top CD instruments. Recent applications and latest beamline upgrades will be discussed. 2812-PosWidefield Multi-Frequency Fluorescence Lifetimeimaging using a Two-Tap CMOS Camera Withlateral Electric Field Charge Modulators

Metrics & MoreArticle RecommendationsI n this erratum, we are submitting the corrected Figure 5, as provided below. The corrected version has been approved by coauthors.

melanocyte development and differentiation. MITF has also been identified as a lineage-specific oncoprotein in melanoma, and knockdown of MITF function results in the activation of cellular senescence. MITF contains N-terminal and C-terminal activation domains as well as a central basic helix-loop-helix DNA binding motif. Both the N-terminal and C-terminal activation domains directly interact with the homologous histone acetyltransferases CBP/p300 and are required for MITF-dependent transcriptional activation. We have used a combination of pull-down experiments, NMR spectroscopy, biophysical studies, and functional transactivation assays to show that the N-terminal activation domain of MITF is intrinsically disordered in solution and directly interacts with the TAZ2 domain of CBP/p300 with high affinity. NMR-based titrations indicate that MITF also interacts with the KIX and TAZ1 domains of CBP/p300, raising the possibility of co-operative interactions between these domains. Competition experiments indicate that E1A is able to displace MITF from TAZ2. Experiments aimed at determining the functional effect of blocking the MITF:TAZ2 interaction are ongoing. These results provide insight as to how MITF may control gene expression in melanoma and support a model in which MITF interacts with multiple domains of CBP/p300 to activate transcription of MITF-target genes.