Molecular Brightness Approach for FRET Analysis of Donor-Linker-Acceptor Constructs at the Single Molecule Level: A Concept.

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.

Fluorescence depolarization dynamics of ionic strength sensors using time-resolved anisotropy.

Eukaryotic cells exploit dynamic and compartmentalized ionic strength to impact a myriad of biological functions such as enzyme activities, protein-protein interactions, and catalytic functions. Herein, we investigated the fluorescence depolarization dynamics of recently developed ionic strength biosensors (mCerulean3-linker-mCitrine) in Hofmeister salt (KCl, NaCl, NaI, and Na2SO4) solutions. The mCerulean3-mCitrine acts as a Förster resonance energy transfer (FRET) pair, tethered together by two oppositely charged α-helices in the linker region. We developed a time-resolved fluorescence depolarization anisotropy approach for FRET analyses, in which the donor (mCerulean3) is excited by 425-nm laser pulses, followed by fluorescence depolarization analysis of the acceptor (mCitrine) in KE (lysine-glutamate), arginine-aspartate, and arginine-glutamate ionic strength sensors with variable amino acid sequences. Similar experiments were carried out on the cleaved sensors as well as an E6G2 construct, which has neutral α-helices in the linker region, as a control. Our results show distinct dynamics of the intact and cleaved sensors. Importantly, the FRET efficiency decreases and the donor-acceptor distance increases as the environmental ionic strength increases. Our chemical equilibrium analyses of the collapsed-to-stretched conformational state transition of KE reveal that the corresponding equilibrium constant and standard Gibbs free energy changes are ionic strength dependent. We also tested the existing theoretical models for FRET analyses using steady-state anisotropy, which reveal that the angle between the dipole moments of the donor and acceptor in the KE sensor are sensitive to the ionic strength. These results help establish the time-resolved depolarization dynamics of these genetically encoded donor-acceptor pairs as a quantitative means for FRET analysis, which complement traditional methods such as time-resolved fluorescence for future in vivo studies.

FRET Analysis of Ionic Strength Sensors in the Hofmeister Series of Salt Solutions Using Fluorescence Lifetime Measurements.

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.

Effects of random motion in traveling and grazing herds.

We examine the role that randomness or noise in individual motion may play in forming effective grazing strategies for herd members as they collectively move toward a destination. Through a model where animals are attracted to Voronoi neighbors as well as a destination endpoint, we show that including a significant random motion component can speed up the movement of a herd toward this destination, increase the efficiency that food is acquired during the travel, and facilitate a natural herd shape that mitigates predation risk. Specifically, if the influence of the Voronoi neighbors on individual motion is equal to the pull toward the destination, we find that optimal travel time and food consumption efficiency occurs for noise approximately twice as strong as the influence of herd members to each other, in a range of herd sizes from 10 to 100. We find that reducing the destination influence lowers this optimal noise only slightly, with random motion still exceeding the influence of neighbors. For a destination influence exceeding that of the Voronoi neighbors, an additional travel mode appears with minimal noise and aligned velocities in which the herd marches directly toward the endpoint. Our results are consistent with observational evidence of random motion in several animal groups, and motivate its generalization to traveling and grazing herds.