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.

The heterogeneous cellular environment influences a myriad of biological processes. For example, macromolecular crowding affects biochemical reactions, protein-protein interactions, and protein folding. Additionally, the structure-function relationship of biomolecules and enzymatic activities are sensitive to the surrounding ionic strength. In this contribution, we highlight our recent studies on a family of donor-linkeracceptor constructs, which were designed for mapping the macromolecular crowding and ionic strength in living cells. Integrated ultrafast laser spectroscopy methods have been employed to quantify the Förster resonance energy transfer (FRET) and the donor-acceptor distance as a measure of the sensitivity of these constructs to environmental changes. The donor-acceptor FRET pairs are intrinsically fluorescent cyan and yellow proteins, respectively, that can be genetically encoded in living cells. The sensitivity of these constructs to environmental biomimetic crowding and ionic strength was investigated as a function of the sequence and charge of the linker regions, as well as the identity of the donor protein. Integrating noninvasive, quantitative laser-induced fluorescence methods with FRET, as a molecular ruler, provides a powerful tool for cellular studies towards mapping out macromolecular crowding and ionic strength in living cells. Our results are key for the development of rational design strategies for engineering enhanced noninvasive biosensors with better environmental sensitivities. The same sensors were used as a model system for developing new experimental approaches for protein-protein interaction and FRET studies. Importantly, these diagnostic molecular and analytical tools set the stage for understanding the correlation between these environmental factors and cellular functions.

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.