Rhodamine spirolactams (RSLs) have recently emerged as popular fluorescent pH probes due to their fluorescence turn-on capability and ease of functionalization at the spirolactam nitrogen. Design of RSLs is often driven by biological targeting or compatibility concerns, rather than the pH sensitivity of the probe, and the relationship between RSL structure and pK is not well understood. To elucidate the relationship between pK values and the properties of substituents attached to the spirolactam nitrogen, a series of 19 aniline-derived RSLs is presented. RSLs derived from di-ortho-substituted anilines exhibit pK tunability across the moderately acidic region (ca. pH 4-6). Evaluation of pK data using the Fujita-Nishioka model for ortho substituent effects reveals that both steric and electronic substituent properties influence RSL pH responsiveness, with pK values increasing as substituent size and electron withdrawing character increase. These trends are attributed to changes in the RSL structure induced by large substituents, and to electronic influences on the protonated spirocyclic reaction intermediate. To demonstrate the practical applicability of these probes in completely aqueous environments, RSL-doped conjugated polymer nanoparticles that exhibit a ratiometric fluorescence response to changing pH levels are presented.
Fluorescent proteins (FPs) have revolutionized cell biology by allowing genetic tagging of specific proteins inside living cells. In conjunction with Fö rster's resonance energy transfer (FRET) measurements, FP-tagged proteins can be used to study protein-protein interactions and estimate distances between tagged proteins. FRET is mediated by weak Coulombic dipole-dipole coupling of donor and acceptor fluorophores that behave independently, with energy hopping discretely and incoherently between fluorophores. Stronger dipole-dipole coupling can mediate excitonic coupling in which excitation energy is distributed near instantaneously between coherently interacting excited states that behave as a single quantum entity. The interpretation of FP energy transfer measurements to estimate separation often assumes that donors and acceptors are very weakly coupled and therefore use a FRET mechanism. This assumption is considered reasonable as close fluorophore proximity, typically associated with strong excitonic coupling, is limited by the FP b-barrel structure. Furthermore, physiological temperatures promote rapid vibrational dephasing associated with a rapid decoherence of fluorophore-excited states. Recently, FP dephasing times that are 50 times slower than traditional organic fluorophores have been measured, raising the possibility that evolution has shaped FPs to allow stronger than expected coupling under physiological conditions. In this study, we test if excitonic coupling between FPs is possible at physiological temperatures. FRET and excitonic coupling can be distinguished by monitoring spectral changes associated with fluorophore dimerization. The weak coupling mediating FRET should not cause a change in fluorophore absorption, whereas strong excitonic coupling causes Davydov splitting. Circular dichroism spectroscopy revealed Davydov splitting when the yellow FP Venus A206 dimerizes, and a novel approach combining photon antibunching and fluorescence correlation spectroscopy was used to confirm that the two fluorophores in a Venus A206 homodimer behave as a single-photon emitter. We conclude that excitonic coupling between Venus A206 fluorophores is possible at physiological temperatures.
In a two-state molecular system, transition paths comprise the portions of trajectories during which the system transits from one stable state to the other. Because of their low population, it is essentially impossible to obtain information on transition paths from experiments on a large sample of molecules. However, single-molecule experiments such as laser optical tweezers or Förster resonance energy transfer (FRET) spectroscopy have allowed transition-path durations to be estimated. Here, we use molecular simulations to test the methodology for obtaining information on transition paths in single-molecule FRET by generating photon trajectories from the distance trajectories obtained in the simulation. Encouragingly, we find that this maximum likelihood analysis yields transition-path times within a factor of 2–4 of the values estimated using a good coordinate for folding, but tends to systematically underestimate them. The underestimation can be attributed partly to the fact that the large changes in the end–end distance occur mostly early in a folding trajectory. However, even if the transfer efficiency is a good reaction coordinate for folding, the assumption that the transition-path shape is a step function still leads to an underestimation of the transition-path time as defined here. We find that allowing more flexibility in the form of the transition path model allows more accurate transition-path times to be extracted and points the way toward further improvements in methods for estimating transition-path time and transition-path shape.
Microtubules are hollow protein filaments consisting of the alpha/beta-tubulin subunit, and they play important roles in various biological processes such as cell division, intracellular transport, cell motility and cell morphogenesis. The dynamics of microtubules is critical to the proper function of microtubules in cell division. One of the challenges in improving our understanding of microtubule dynamics is the small size of tubulin subunits. Because each subunit is only few nanometers in size and significantly smaller than the wavelength of light, optical microscopy cannot be used to resolve the interactions of the subunits, which lead to the formation of microtubules, in real time. We developed an in vitro spectroscopy assay for detecting microtubule formation below the diffraction limit of light. The assay is based on Förster resonance energy transfer between fluorescent molecules of a single type (homoFRET). Our results indicate that homoFRET can be used to detect short microtubules even when they are diffraction limited (smaller than few hundred nanometers). We also demonstrate that when fluorophores with appropriate Förster distance are used, this technique can be highly sensitive to the formation of microtubules but less sensitive to the extent of microtubule elongation, making it suitable for detecting microtubule nucleation.
Biological processes performed by proteins interacting with and processing DNA and RNA are key to cell metabolism and life. Detailed insights into these processes provide essential information for understanding the molecular basis of life and the pathological conditions that develop when such processes go awry. The next scientific breakthrough consists in the actual, direct, real-time observations and measurements of the individual mechanisms involved, in order to validate and complete the current biological models. Single-molecule technologies offer an exciting opportunity to meet these challenges and to study protein function and activity in real-time and at the single-molecule level. Here, we present our efforts for further enabling discoveries in the field of biology and biophysics using both the combination of optical tweezers with singlemolecule fluorescence microscopy (C-Trap TM ). We show the latest applications of these technologies that can enhance our understanding not only in the field of DNA/RNA-protein interactions but also in the fields of molecular motors, protein folding/unfolding, cell membranes and genome structure and organization. These experiments show that the technological advances in hybrid single-molecule methods can be turned into an easy-to-use and stable instrument that has the ability to open up new venues in many research areas.
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