Spectral characteristics of fluorophores formed via interaction between all-trans-retinal with rhodopsin and lipids in photoreceptor membrane of retina rod outer segments

It is shown that all-trans-retinal under model conditions of its excessive accumulation in photoreceptor membranes interacts with amino groups of rhodopsin and lipids, forming at least three distinct fluorophores with fluorescence quantum yield 20–40 times higher than that of free all-trans-retinal. These retinal derivatives are likely precursors of photo- and cytotoxic fluorophores of lipofuscin and in particular of A2E. Spectral characteristics of fluorophores have been described. Picosecond time-resolved laser fluorescence spectroscopy was used to study kinetics of fluorescence decay of both free and bound all-trans-retinal; fluorophores were determined and their lifetimes have been measured. Based on calculations it is shown that the decay kinetics of all-trans-retinal derivatives consists of three components with lifetimes equal to 48, 208, and 900 ps; kinetics of free all-trans-retinal is monoexponential with lifetime of 31 ps. The chemical nature of fluorophores with the lifetimes obtained is discussed.     

Model of the pathway of −1 frameshifting: Long pausing

It has been characterized that the programmed ribosomal ?1 frameshifting often occurs at the slippery sequence on the presence of a downstream mRNA pseudoknot. In some prokaryotic cases such as the dnaX gene of Escherichia coli, an additional stimulatory signal—an upstream, internal Shine–Dalgarno (SD) sequence—is also necessary to stimulate the efficient ?1 frameshifting. However, the molecular and physical mechanism of the ?1 frameshifting is poorly understood. Here, we propose a model of the pathway of the ?1 translational frameshifting during ribosome translation of the dnaX ?1 frameshift mRNA. With the model, the single-molecule fluorescence data (Chen et al. (2014) [29]) on the dynamics of the shunt either to long pausing or to normal translation, the tRNA transit and sampling dynamics in the long-paused rotated state, the EF-G sampling dynamics, the mean rotated-state lifetimes, etc., are explained quantitatively. Moreover, the model is also consistent with the experimental data (Yan et al. (2015) [30]) on translocation excursions and broad branching of frameshifting pathways. In addition, we present some predicted results, which can be easily tested by future optical trapping experiments.     

Phase-sensitive fluorescence spectroscopy: A new method to resolve fluorescence lifetimes or emission spectra of components in a mixture of fluorophores

A novel phase fluorometric method is described which permits direct recording of individual emission spectra from a mixture of two flourescent compounds. Additionally, the lifetimes of each component may be determined by examination of the phase-sensitive fluorescence spectra. The method utilizes phase-sensitive detection of the sinusoidally modulated emission from a phase fluorometer. Resolution of the individual emission spectra in the mixture requires different fluorescence lifetimes for each components. Determination of the individual lifetime requires knowledge of the steady-state emission spectra of the components. Use of low-frequency (≈ 10 Hz) cross-correlated signals eliminates the need for high-frequency frequency (≈10⁶ Hz) phase-sensitive detection. A mixture of 2-p-toluidinyl-6-naphthalenesulfonic acid (TNS) and 6-propionyl-2-(dimethylamino)naphthalene (PRODAN) was used to demonstrate the possibility of phase resolution of fluorophore mixture and to confirm theoretical predictions. A mixture of dibenzo[a,h]anthracene and dibenzo[c,g]carbazole was used to demonstrate that phase resolution is possible for spectra which overlap strongly and which are highly structured. In addition, the possibility of using phase-sensitive emission spectra for the resolution of excited-state reactions was demonstrated with anthracene and its diethylaniline exciplex. From a sample whose steady-state emission displayed both components we directly recorded the emission spectrum of anthracene monomer and the exciplex. For all these samples the dependence of the individual intensities on the phase angle of the detector agreed precisely with that expected on the basis of the individual fluorescence lifetimes. The detector phase angles chosen for suppression of each component in the mixture also agreed with the measured lifetimes. Thus, phase-sensitive fluorescence spectra can reveal individual spectral distributions or lifetimes. This method will be useful in the analysis fluorescence emissions which frequently occur from proteins, membranes and other biological samples.     

Time-resolved measurements of fluorescence from reaction centres of Rhodopseudomonas viridis and the effect of menaquinone reduction

The kinetics of the fluorescence emitted by the ‘special pair’ of bacteriochlorophyll b molecules in reaction centres from Rhodopseudomonas viridis was recorded in the near infrared, with a time resolution of 1 ns. In nonreduced reaction centres two decay components were resolved with lifetimes of <0.5 and 2.5 ns. Upon reduction of the menaquinone electron acceptor three decay components were detected with lifetimes of < 0.5, 2.5 and 15ns.     

Excited‐state lifetime studies of the three tryptophan residues in the N‐lobe of human serum transferrin

The energy transfer from the three Trp residues at positions 8, 128, and 264 within the human serum transferrin (hTF) N-lobe to the ligand to metal charge transfer band has been investigated by monitoring changes in Trp fluorescence emission and lifetimes. The fluorescence emission from hTF N-lobe is dominated by Trp264, as revealed by an 82% decrease in the quantum yield when this Trp residue is absent. Fluorescence lifetimes were determined by multifrequency phase fluorometry of mutants containing one or two Trp residues. Decays of these samples are best described by two or three discrete lifetimes or by a unimodal Lorentzian distribution. The discrete lifetimes and the center of the lifetime distribution for samples containing Trp128 and Trp264 are affected by iron. The distribution width narrows on iron removal and is consistent with a decrease in dynamic mobility of the dominant fluorophore, Trp264. Both the quantum yield and the lifetimes are lower when iron is present, however, not proportionally. The greater effect of iron on quantum yields is indicative of nonexcited state quenching, i.e., static quenching. The results of these experiments provide quantitative data strongly suggesting that Förster resonance energy transfer is not the sole source of Trp quenching in the N-lobe of hTF.     

Long-Lived, High-Strength States of ICAM-1 Bonds to β2 Integrin, II: Lifetimes of LFA-1 Bonds Under Force in Leukocyte Signaling

Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant α_Lβ₂ immobilized on microspheres and β₂ integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from β₂ integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant α_Lβ₂ on microspheres in millimolar solutions of divalent cations (Ca²⁺, Mg²⁺, Mn²⁺). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to β₂ integrin on the cell surface. Even though ICAM-1 bonds to recombinant α_Lβ₂ on microspheres in Mg²⁺ or Mn²⁺ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg²⁺ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg²⁺ or Mn²⁺ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bond-like statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.     

Effect of cholinergic ligands and local anesthetics on acetylcholine receptor enriched membrane preparations from Torpedo californica electroplax.

Pyrene was introduced in acetylcholine receptor (AcChR)-rich membrane preparations of Torpedo californica electroplax. The lifetime of the singlet excited state of pyrene was used to probe the properties of the hydrocarbon regions of the lipid bilayer as well as the possible perturbing effects of cholinomimetic agents on this region. After excitation with a single 15-ns pulse with a Q-switched ruby laser, the lifetime of the pyrene singlet excited state in the membranes was 200 ns. In desensitized membranes the pyrene fluorescence lifetimes remained unchanged when the cholinergic ligands carbamylcholine, d-tubocurarine, decamethonium, and hexamethonium, as well as α-bungarotoxin, were present. By contrast, the lifetime was shortened when local anesthetics were present. In sensitized membranes no changes in the pyrene lifetimes were detected when the membranes were converted from their resting state to a carbamylcholine-induced “desensitized state.” Water-soluble fluorescence quenchers affected the lifetime of pyrene in membranes. The second order rate constants for the pyrene-quencher interaction were used to detect changes in fluidity and/or membrane lipid accessibility to quenchers induced by ligands or anesthetics. No changes were detected in the quenching constants of nitromethane or Tl⁺ in the presence of cholinergic agents (with the exception of d-tubocurarine); on the other hand, a marked decrease in Tl⁺ accessibility was induced by the anesthetics procaine and tetracaine. Fluorescene dynamics measurements indicate that the hydrocarbon core of the bulk lipid in electroplax is not significantly affected by binding cholinergic ligands to membranebound AcChR. However, the hydrophobic region of the membrane is perturbed by both local anesthetics and one cholinergic ligand, d-tubocurarine. Pyrene was also incorporated into lipid vesicles prepared from T. californica electroplax lipids. The fluorescence lifetimes and quenching values of these lifetimes yielded results similar to those obtained with both sensitized and “desensitized” membrane preparations. The d-tubocurarine effect on the Tl⁺ quenching of the pyrene probe is ascribed to direct interaction of d-tubocurarine with the lipids. These findings favor a mechanism in which perturbation of the hydrophobic (lipid) environment of the AcChR in membranes by local anesthetics and even d-tubocurarine may influence the receptor conversion: sensitized state ? desensitized state.     

Fluorescence spectroscopic studies of pyrene-actin adducts

Reaction kinetic studies of the sulfhydryl-directed fluorescent probes N-(1-pyrene)maleimide (PM) and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle showed that there were three accessible sulfhydryl groups in actin. Fluorescence spectral studies showed energy transfer from aromatic amino acid residues to fluorophore reacted at Cys-373, as well as weak excimer fluorescence probably due to doubly labeled molecules at Cys-10 and Cys-373. These results provide further evidence that trytophan and tyrosine residues are located near the probe attached to Cys-373 or Cys-10 and the latter two thiols are in close proximity. In aged PM-Iabeled F-actin, the succinimido ring of PM underwent intramolecular aminolysis. resulting in large emission spectral changes and increased excimer fluorescence. Solvent perturbation studies indicate that the probes were located in a hydrophobic environment; their quantum yield and spectrum properties were very sensitive to changes in the microenvironment. Nanosecond-pulse fluorimetry studies revealed complex fluorescence emission decays with three intrinsic lifetimes in adducts with low molecular weight thiols as well as in labeled proteins. Fluorescence lifetimes were 17. 48 and 111 ns for the pyrenemaleimide adduct of actin, and 3, 14 and 60 ns for the pyrenyliodoacetamide adduct. Supporting evidence is given for the argument that multiple fluorescence lifetimes are an intrinsic property of the pyrene derivatives and are not due to the presence of impurity or heterogeneity in the protein reaction sites. Because of their high sensitivity and long lifetimes, pyrene derivatives are extremely useful.     

Lifetimes of mRNA molecules directing the synthesis of viral proteins in herpes simplex virus-infected cells.

Possible variations in the functional lifetimes of herpes simplex virus type 1 mRNA molecules in infected HeLa cells were studied. As shown by the rate of decrease of radioactive amino acid incorporation into viral proteins after the addition of actinomycin, the average lifetime of early viral mRNA's are shorter than those for the late messenger species. In addition, when the viral proteins made after the addition of actinomycin were further analyzed by gel electrophoresis, it was found that messengers for individual viral proteins translated within the early or late time period also had some differences in their functional lifetimes. These results indicate that the synthesis of herpes simplex virus type 1 proteins during the replicative cycle is regulated in part by mechanisms controlling the functional lifetimes of viral mRNA's.     

In Situ Activity of Suspended and Immobilized Microbial Communities as Measured by Fluorescence Lifetime Imaging

In this study, the feasibility of fluorescence lifetime imaging (FLIM) for measurement of RNA:DNA ratios in microorganisms was assessed. The fluorescence lifetime of a nucleic acid-specific probe (SYTO 13) was used to directly measure the RNA:DNA ratio inside living bacterial cells. In vitro, SYTO 13 showed shorter fluorescence lifetimes in DNA solutions than in RNA solutions. Growth experiments with bacterial monocultures were performed in liquid media. The results demonstrated the suitability of SYTO 13 for measuring the growth-phase-dependent RNA:DNA ratio in Escherichia coli cells. The fluorescence lifetime of SYTO 13 reflected the known changes of the RNA:DNA ratio in microbial cells during different growth phases. As a result, the growth rate of E. coli cells strongly correlated with the fluorescence lifetime. Finally, the fluorescence lifetimes of SYTO 13 in slow- and fast-growing biofilms were compared. For this purpose, biofilms developed from activated sludge were grown as autotrophic and heterotrophic communities. The FLIM data clearly showed a longer fluorescence lifetime for the fast-growing heterotrophic biofilms and a shorter fluorescence lifetime for the slow-growing autotrophic biofilms. Furthermore, starved biofilms showed shorter lifetimes than biofilms supplied with glucose, indicating a lower RNA:DNA ratio in starved biofilms. It is suggested that FLIM in combination with SYTO 13 represents a useful tool for the in situ differentiation of active and inactive bacteria. The technique does not require radioactive chemicals and may be applied to a broad range of sample types, including suspended and immobilized microorganisms.     

Electrostatic Interactions in the Binding Pathway of a Transient Protein Complex Studied by NMR and Isothermal Titration Calorimetry

Much of our knowledge of protein binding pathways is derived from extremely stable complexes that interact very tightly, with lifetimes of hours to days. Much less is known about weaker interactions and transient complexes because these are challenging to characterize experimentally. Nevertheless, these types of interactions are ubiquitous in living systems. The combination of NMR relaxation dispersion Carr–Purcell–Meiboom–Gill (CPMG) experiments and isothermal titration calorimetry allows the quantification of rapid binding kinetics for complexes with submillisecond lifetimes that are difficult to study using conventional techniques. We have used this approach to investigate the binding pathway of the Src homology 3 (SH3) domain from the Fyn tyrosine kinase, which forms complexes with peptide targets whose lifetimes are on the order of about a millisecond. Long range electrostatic interactions have been shown to play a critical role in the binding pathways of tightly binding complexes. The role of electrostatics in the binding pathways of transient complexes is less well understood. Similarly to previously studied tight complexes, we find that SH3 domain association rates are enhanced by long range electrostatics, whereas short range interactions are formed late in the docking process. However, the extent of electrostatic association rate enhancement is several orders of magnitudes less, whereas the electrostatic-free basal association rate is significantly greater. Thus, the SH3 domain is far less reliant on electrostatic enhancement to achieve rapid association kinetics than are previously studied systems. This suggests that there may be overall differences in the role played by electrostatics in the binding pathways of extremely stable versus transient complexes.     

Excimer fluorescence of pyrene-tropomyosin adducts

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct: and about 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35 and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the micro-environment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.     

Dependence of the energy lifetime on the type of anomalous losses in stellarators

A relatively simple model of transport process in stellarators that was proposed earlier by the author on the basis of neoclassical theory makes it possible to determine the density and temperature profiles of the plasma components, the ambipolar electric field profile, and the particle and energy lifetimes from the given device parameters and given particle and energy sources with allowance for anomalous losses. The results of numerical simulations carried out with this model for the L-2M, ATF, CHS, and LHD stellarators over broad ranges of plasma densities and absorbed powers showed that the plasma energy lifetimes in these devices coincide to within factors on the order of two with those found from empirical scalings. A specific model of anomalous losses was chosen for calculations. Results are presented from simulations with a more general form of the anomalous thermal conductivity. Namely, the thermal conductivity is chosen to be K _j ^(a) ≈ N ^α T _j ^β B ₀ ^?γ , where N(r) is the plasma density and T _j (r) is the temperature of the jth plasma component (j = e, i). The parameters α, β, and γ are set equal to α = 1, β = 2, and γ = 1; α = 0.5, β = 2.5, and γ = 1; α = 1.5, β = 2, and γ = 2; α = 1, β = 2.5, and γ = 2; and α = 1.5, β = 2.5, and γ = 2. The simulations have been done for the L-2M and LHD stellarators. It is found that, in all the five models, the calculated energy lifetimes τ _c are essentially independent of the functional form of the anomalous thermal conductivity and coincide to within a factor on the order of two with those following from the LHD scaling.     

Heterogeneity of the two tryptophanyl residues in the lac repressor of Escherichia coli.

The wild-type lac repressor of Escherichia coli is a tetrameric protein which contains two tryptophanyl residues per subunit at positions 190 and 209. Solute perturbation studies of the tryptophan fluorescence of the repressor were performed using a polar but uncharged quencher, acrylamide, to prevent possible bias caused by ionic quenchers. The results indicate that the two tryptophan residues have different accessibilities to the quencher. In addition, contrary to a previous report, the accessibility of these tryptophan residues is not altered by isopropyl-β-d-thiogalactoside (IPTG) binding to the repressor. Similar studies with mutant lac repressor containing only a single tryptophan either at positions 190 or 209 suggest that tryptophan 209 is located in a region which is perturbed by inducer binding. That the two tryptophanyl residues have heterogeneous environments was further confirmed by nanosecond fluorescence spectroscopy which showed the wild-type lac repressor exhibiting two excited-state lifetimes, τ₁ = 5.3 ns and τ₂ = 10 ns. In the presence of 10^?3m IPTG, only a single lifetime of 6 ns was observed for the wild-type repressor suggesting that the inducer perturbs the tryptophan residue with the longer lifetime but not the one with the shorter lifetime. This is in accord with the observation that the mutant repressor containing only tryptophan 190 (the Tyr-209 repressor) has a single lifetime of 4.5 ns which is not altered by IPTG binding. The surprising finding that the mutant repressor which contains only tryptophan 209 (the Tyr-190 repressor) shows two excited-state lifetimes has been interpreted to indicate that the repressor either does not exhibit fourfold symmetry in its subunit arrangement or is present in two different conformational states.     

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

Planar lipid bilayers suspended in apertures provide a controlled environment for ion channel studies. However, short lifetimes and poor mechanical stability of suspended bilayers limit the experimental throughput of bilayer electrophysiology experiments. Although bilayers are more stable in smaller apertures, ion channel incorporation through vesicle fusion with the suspended bilayer becomes increasingly difficult. In an alternative bilayer stabilization approach, we have developed shaped apertures in SU8 photoresist that have tapered sidewalls and a minimum diameter between 60 and 100 μm. Bilayers formed at the thin tip of these shaped apertures, either with the painting or the folding method, display drastically increased lifetimes, typically >20 h, and mechanical stability, being able to withstand extensive perturbation of the buffer solution. Single-channel electrical recordings of the peptide alamethicin and of the proteoliposome-delivered potassium channel KcsA demonstrate channel conductance with low noise, made possible by the small capacitance of the 50 μm thick SU8 septum, which is only thinned around the aperture, and unimpeded proteoliposome fusion, enabled by the large aperture diameter. We anticipate that these shaped apertures with micrometer edge thickness can substantially enhance the throughput of channel characterization by bilayer lipid membrane electrophysiology, especially in combination with automated parallel bilayer platforms.