Time-resolved Linear Dichroism Research Articles

Microviscosity in E. coli Cells from Time-Resolved Linear Dichroism Measurements

A protein's folding or function depends on its mobility through the viscous environment that is defined by the presence of macromolecules throughout the cell. The relevant parameter for this mobility is microviscosity-the viscosity on a time and distance scale that is important for protein folding/function movements. A quasi-null, ultrasensitive time-resolved linear dichroism (TRLD) spectroscopy is proving to be a useful tool for measurements of viscosity on this scale, with previous in vitro studies reporting on the microviscosities of crowded environments mimicked by high concentrations of different macromolecules. This study reports the microviscosity experienced by myoglobin in the E. coli cell's heterogeneous cytoplasm by using TRLD to measure rotational diffusion times. The results show that photolyzed deoxyMb ensembles randomize through environment-dependent rotational diffusion with a lifetime of 34 ± 6 ns. This value corresponds to a microviscosity of 2.82 ± 0.42 cP, which is consistent with previous reports of cytoplasmic viscosity in E. coli. The results of these TRLD studies in E. coli (1) provide a measurement of myoglobin mobility in the cytoplasm, (2) taken together with in vitro TRLD studies yield new insights into the nature of the cytoplasmic environment in cells, and (3) demonstrate the feasibility of TRLD as a probe of intracellular viscosity.

Time-Resolved Linear Dichroism Measurements of Carbonmonoxy Myoglobin as a Probe of the Microviscosity in Crowded Environments.

The distribution of viscosities in living cells is heterogeneous because of the different sizes and natures of macromolecular components. When thinking about protein folding/function processes in such an environment, the relevant (micro)viscosity at the micrometer length scale is necessarily distinguished from the bulk (macro)viscosity. The concentration dependencies of microviscosities are determined by a number of factors, such as electrostatic interactions, van der Waals forces, and excluded volume effects. To explore such factors, the rotational diffusion time of myoglobin in the presence of varying concentrations of macromolecules that differ in molecular weight (dextran 6000, 10 000, and 70 000), shape (dextran versus Ficoll), size, and surface charge is measured with time-resolved linear dichroism spectroscopy. The results of these studies offer simple empirically determined linear and exponential functions useful for predicting microviscosities as a function of concentration for these macromolecular crowders that are typically used to study crowding effects on protein folding. To understand how relevant these microviscosity measurements are to intracellular environments, the TRLD results are discussed in the context of studies that measure viscosity in cells.

High-aperture-excitation and -detection fluorescence spectroscopy with polarized light

A description of fluorescence depolarization spectroscopy in the case of high-aperture-detection and -excitation is considered and described in spherical representation. The formalism outlined applies to one- and two-photon-excitation polarized fluorescence spectroscopy for different kinds of molecular media, including thin layers of organized molecular assemblies, solutions and interfaces. It applies to standard fluorescence and time-resolved linear dichroism measurements, fluorescence microscopy and evanescent-wave-excitation fluorescence experiments.

Influence of Localized Excited States on the Transition Moment Directions of Charge Transfer Complex Absorptions

The influence of localized excited (LE) states on the spectroscopy of charge transfer (CT) complexes has been examined for a series of complexes formed between methyl-substituted benzene donors and 1,2,4,5-tetracyanobenzene as acceptor in 1,2-dichloroethane and octanenitrile solvents. A molecular orbital model was used to describe the appearance of multiple CT absorption bands that occur in the spectra of these complexes. The influence of LE states in these CT absorptions was explored using time-resolved linear dichroism spectroscopy where the direction of the CT transition moment vector (TMV) was used to probe the magnitude of intensity borrowing. The TMV directions for each of the observed CT transitions within the absorption spectra were determined for several complexes. In some cases, the observed CT transitions were interpreted as being pure CT transitions; in others the observed transitions are influenced significantly by a LE transition. The correlation between the TMV directions and the transition energy suggests that the magnitude of intensity borrowing is influenced not only by the energy difference between the CT and LE transitions but also by the specific character of the transitions under consideration.

Effect of Digitonin on the Rhodopsin Meta I - Meta II Equilibrium

Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Effect of Digitonin on the Rhodopsin Meta I–Meta II Equilibrium¶

Absorbance difference spectra were recorded from 10 micros to 540 ms after photoexcitation of sonicated suspensions of hypotonically washed bovine rod outer segments with varying amounts of the detergent digitonin added (0 to 2%) at 20 degrees C. Metarhodopsin I480 and metarhodopsin II displayed the expected anomalous pH dependence at pH 6 and 8 (i.e. opposite to that expected from direct protonation of the chromophore Schiff base). However, increasing levels of digitonin eliminated the pH dependence of the equilibrium, and at 2% digitonin the pH 6 and pH 8 data were both similar to the data collected at pH 8 without digitonin. Addition of 0.5% azolectin restored approximately 50% of the anomalous pH dependence at pH 6 in the 2% digitonin sample. The possibility that digitonin induced large-scale aggregation of rhodopsin in the disk membrane that could be reversed by azolectin was tested using time-resolved linear dichroism. Those results showed that even 0.3% digitonin disrupted the membrane, and no large aggregates were detectable under any conditions. Thus, digitonin reduces the activity of a component of the disk membrane required for metarhodopsin II formation, and that deficiency can be compensated for by azolectin.

Time-Resolved Linear Dichroism and Linear Birefringence of Bacteriorhodopsin at Alkaline pH: Identification of Two N Substates with Different Orientations of the Transition Dipole Moment

Transient linear dichroism and linear birefringence changes in the photocycle of bacteriorhodopsin at alkaline pH were measured with magnetically oriented purple membrane samples using the method of isotropic excitation (Borucki, B.; Otto, H.; Heyn, M. P. J. Phys. Chem. B 1999, 103, 6371−6383). At pH 10.4, the accumulation of the O intermediate is negligible, and N is the only intermediate that is relevant in the M decay and the recovery of the bR ground state. We introduced a new analytical approach that is model-independent and makes use of the transient linear dichroism in combination with isotropic absorbance changes. SVD analysis reveals that only one species (spectral component), namely, the N intermediate, is involved in the M decay at pH 10.4. The spectrum of this intermediate and its average anisotropy are determined from an eigenvalue equation. The transient linear dichroism data suggest a transition between two substates of the N intermediate with different anisotropies (i.e., with different orientations of the electronic transition dipole moment). Assuming that the polar angle of the transition dipole moment with respect to the membrane normal is 70° in the bR ground state, we get 66.3° (upper limit) for the first and 67.9° (lower limit) for the second N substate. The reorientation of the chromophore in the N1 → N2 transition is probably associated with the movement of the protonated Schiff base away from Asp96, suggesting that it serves as a reprotonation switch for Asp96 by stabilizing the protonation state of the Schiff base. In contrast to measurements at neutral pH, the linear birefringence changes at alkaline pH contain a wavelength-independent component that is not due to the transient absorption changes and whose time dependence is correlated with the kinetics of the N intermediate. This result is attributed to an electro-optical effect caused by the large electric field generated by the transient negative charge on Asp96 in the N intermediate. Alternatively this change in Δn may be due to the major conformational change in the cytoplasmic region of the membrane during N.

Application of time-resolved linear dichroism spectroscopy: Intensity borrowing in charge transfer complex absorption spectra

Time-resolved linear dichroism spectroscopy has been used to study the influence of solvent on the charge transfer complex formed between hexamethylbenzene and 1,2,4,5-tetracyanobenzene. It was shown that cyano-substituted solvents induce a 1500 cm–1 increase in the charge transfer transition energies relative to those observed in chlorinated solvents. Furthermore, the angle between the charge transfer absorption transition moments and the photochemically produced radical anion absorption transition moment, after relaxation, has been measured for this complex in several solvents. A simple model was used to correlate the angles measured using time-resolved linear dichroism spectroscopy with the extent of localized excitation mixed into the charge transfer transitions. These measurements reveal that different charge transfer transitions borrow intensity from the localized excitation to different extents. By using different excitation wavelengths, the partitioning of the borrowed intensity among the charge transfer transitions of this complex could be evaluated for the first time.Key words: 1,2,4,5-tetracyanobenzene, hexamethylbenzene, donor–acceptor complex, photoinduced electron transfer, photoselection.

Application of Time-Resolved Linear Dichroism Spectroscopy: Rapid Relaxation of Excited Charge Transfer Complexes

Time-resolved linear dichroism spectroscopy has been used to study the charge-transfer complexes formed between methylated benzene donors and 1,2,4,5-tetracyanobenzene as acceptor. The angle between the charge-transfer absorption transition moment and the photochemically produced radical anion absorption transition moment, after relaxation, has been measured for several complexes. A simple theoretical model was used to interpret the measured values. This model correlated the extent of localized excitation mixed into the charge-transfer transitions with the results of time-resolved linear dichroism measurements. Furthermore, the comparison between the measured and predicted transition moment directions suggests that a minor geometry change occurs after excitation of these complexes. The observation of the relative transition moment vector of the initial state(s) for some of the complexes was also possible. In these cases, the initial transition moment vectors observed are not consistent with absorption due...

Application of Time-Resolved Linear Dichroism Spectroscopy: Relaxation of Excited Hexamethylbenzene/1,2,4,5-Tetracyanobenzene Charge-Transfer Complexes

The charge-transfer complex formed between hexamethylbenzene and 1,2,4,5-tetracyanobenzene has been studied using time-resolved linear dichroism spectroscopy. These studies allowed the angle between the transition moment vectors for the absorptions of the ground-state charge-transfer complex and the product contact-radical ion pair to be measured. The results were compared with predictions based on the Mulliken two-state model and an assumed structure of the ground-state complex. The comparison between the observed and predicted angles supports the notion that relaxation of the excited charge-transfer complex leads to a change in the complex geometry. The time dependence of the dichroism allows the average rotational diffusion time constant to be determined. For this complex in 1,2-dichloroethane at room temperature, τOR = 75 ps. This value is comparable to what would be expected based on Debye−Stokes−Einstein diffusion theory at the stick boundary limit.

Microscopic Details of Rotational Diffusion of Perylene in Organic Solvents: Molecular Dynamics Simulation and Experiment vs Debye−Stokes−Einstein Theory

Molecular dynamics simulations and time-resolved linear dichroism measurements have been employed to investigate rotational diffusion of perylene in two organic solvents, cyclohexane, a nonpolar solvent, and 2-propanol, a polar solvent. Both experiments and simulations yield a biexponential rotational anisotropy decay for the long in-plane axis. The calculated time constants were 9 and 44 ps in cyclohexane at 300 K, 13 and 75 ps in 2-propanol at 300 K, and 25 and 126 ps in 2-propanol at 263 K, in excellent agreement with corresponding time-resolved linear dichroism measurements of 14 and 52 ps, 10 and 51 ps, and 22 and 240 ps respectively. Although the viscosity of 2-propanol is more than two times that of cyclohexane at room temperature, the measured rotational reorientation times and the calculated average rotational diffusion coefficients of perylene are similar in the two solvents, demonstrating a breakdown of simple hydrodynamic theory. Analysis of the calculated rotational diffusion coefficients for...

An Application of Time-Resolved Linear Dichroism Spectroscopy: The Excited Singlet State of 1,2:5,6-Dibenzanthracene

Time-resolved linear dichroism spectroscopy allows the relative transition moment vectors of the S1→ Sn, transitions to be obtained. This information can be used to aide in the assignment of the observed excited state and ground state transitions. The transition moment directions are constrained by the symmetry of the absorbing chromophore such that structural changes on the excited state surfaces can also be observed. The time dependence of the dichroism is related to the solution dynamics through the measurement of rotational correlation times. The spectroscopy of l,2:5,6-dibenzanthracene is presented as an example of these measurements.

Reorientation of the Retinylidene Chromophore in the K, L, and M Intermediates of Bacteriorhodopsin from Time-Resolved Linear Dichroism: Resolving Kinetically and Spectrally Overlapping Intermediates of Chromoproteins

We determined the change in orientation of the electronic transition dipole moment with respect to the membrane normal in the K, L, and M intermediates of bacteriorhodopsin using transient linear dichroism. Purple membranes were oriented in a 14 T magnetic field and immobilized in a gel. The oriented purple membranes were excited isotropically and the transient absorbance changes were detected with the sample between two parallel polarizers. The absorbance changes were measured as a function of wavelength, time, and angle between the orientation axis and polarizer direction. In this way, the transient changes in isotropic absorbance, linear dichroism, and linear birefringence were determined with high accuracy. The Kramers−Kronig transform of the transient linear dichroism was in excellent agreement with the transient linear birefringence and this served as a useful control on the reliability of the linear dichroism data. We developed a novel formalism to extract the anisotropies, spectra and time courses...

A Study of the Mechanisms of Slow Religation to Sickle Cell Hemoglobin Polymers Following Laser Photolysis

Time-resolved linear dichroism (TRLD) measurements are conducted on gels of sickle cell hemoglobin following laser photolysis of the carbonyl adduct to monitor religation kinetics to hemoglobin S polymers. The return of the polymer phase to its equilibrium ligation state has been found to be about 1000 times slower than that of the solution phase hemoglobin tetramers. Several mechanisms describing this slow religation to the polymer were proposed: (1) religation occurs through a biomolecular process involving all polymer hemes, (2) religation occurs through a bimolecular process in which only hemoglobin molecules at the polymer ends can participate, and (3) religation occurs through the exchange of ligated hemoglobin molecules in the monomer phase with unligated ones in the polymer phase. To test these mechanisms, measurements are performed on gels having different domain sizes. The results show no relation between domain size and religation kinetics. The independence of religation kinetics and domain size is most consistent with the first of the three mechanisms described above (bimolecular recombination involving all polymer hemes). This result is discussed in terms of a model in which diffusion of the ligand is inhibited in the polymer phase. An understanding of the ligand binding kinetics of sickle hemoglobin polymers could have pathophysiological significance in its relevance to polymer formation and melting during red blood cell circulation.

Reorientations in the bacteriorhodopsin photoscycle are pH dependent

Chromophore reorientations during the bacteriorhodopsin photocycle in the purple membrane of Halobacterium salinarium have been detected by time-resolved linear dichroism measurements of the optical anisotropy over the pH range from 4 to 10 and at ionic strengths from 10 mM to 1 M. The results show that reorientations in the L and M states of bacteriorhodopsin are pH dependent, reaching their largest amplitude when the membrane is at pH 6-8. Reorientations on the millisecond time scale of unexcited spectator proteins in the native purple membrane also depend on pH, consistent with the suggestion that spectator reorientations are triggered by reorientation of the photoexcited protein. The results imply that a group with a PK(a) of 5 to 6 enables reorientations, and that the deprotonation of a site at pH values above 9 restricts reorientational motion. This suggests that reorientations in M may be correlated with proton release.

Chromophore reorientations in the early photolysis intermediates of bacteriorhodopsin

The photoselection-induced time-resolved linear dichroism of a bacteriorhodopsin suspension of purple membrane from 350 to 750 nm is measured by a new pseudo-null measurement technique. In combination with time-resolved absorption measurements, these linear dichroism measurements are used to determine the reorientation of the retinal chromophore of bacteriorhodopsin from 50 ns to 50 microseconds after photolysis. This time range covers the times when the K photointermediate decays to form L, as well as the early times during the formation of the M intermediate in the photocycle. An analysis of the photoselection-induced linear dichroism measured directly, along with the absorbance changes polarized parallel to the linearly polarized excitation, shows that the anisotropy is invariant over this time period, implying that the photolyzed chromophore rotates less than 8 degrees C with respect to unphotolyzed chromophores during this part of the photocycle.

Chromophore Reorientation Relative to the Membrane Plane Detected by Time-Resolved Linear Dichroism during the Bacteriorhodopsin Photocycle in Oriented Purple Membrane

Time-resolved linear dichroism measurements on samples of oriented purple membranes enable reorientations in the plane of the membrane to be distinguished from reorientations with respect to the membrane normal. Our measurements reveal reorientation of the transition dipole of the retinylidene chromophore toward the membrane normal by 14° in the M-state. A smaller reorientation (3°) in the same direction is observed in the L-state and possibly as early as the K-state. The purple membrane was oriented in an 11 T magnetic field and immobilized in a polyacrylamide gel. Time-resolved linear dichroism measurements on purple membrane oriented so that pump and probe beams propagate parallel to the membrane plane are highly sensitive to reorientation with respect to the membrane normal due to the initial orientation of the transition dipole at about 67° with respect to the membrane normal. Measurements where pump and probe beams propagate perpendicular to the membrane plane reveal no significant in-plane reorientation, although the sensitivity of these measurements is not as high. The observed reorientation in the M-state may be related to conformational changes that have been detected in helices C, F, and G.

Polarization-Division Interferometry: Time-Resolved Infrared Vibrational Dichroism Spectroscopy

We report the first direct measurements of time-resolved Fourier transform infrared vibrational dichroism (TR/FT-IR/VD). The central component for these measurements is a polarization-division interferometer (PDI). This interferometer uses an in-house-designed beamsplitter constructed in-house from a BaF2 polarizer and a matching substrate. In conjunction with a linear polarizer in front of the source and two rooftop mirrors, one in each arm of the interferometer, the PDI divides the input beam into two orthogonal linear polarization components, recombines them for interference at the beamsplitter, and directs the output beam at 90° to the direction of input beam. The signals measured as a function of the moving mirror position in the PDI represent the linear/circular dichroism interferograms, whose cosine/sine Fourier transforms yield linear/circular dichroism spectra. Time-resolved dichroism interferograms were measured with the use of this PDI with the asynchronous external perturbation method. Microsecond time-resolved linear dichroism spectra of a nematic liquid crystal, under the influence of bipolar electric field oscillating at 2 kHz, were measured and presented as the first examples.

Reorientations in the bacteriorhodopsin photocycle.

Reversible photoinduced reorientations of bacteriorhodopsin have been detected in suspensions of the purple membrane of Halobacterium salinarium. The anisotropy in bacteriorhodopsin during the nanosecond through millisecond stages of the photocycle was measured by time-resolved linear dichroism and transient absorption measurements. From these measurements the anisotropies of the K, L, M, and O intermediates were determined and related to the chromophore orientation with respect to the initially selected orientation. The anisotropies of the K and L states are 0.38 +/- 0.01 and 0.35 +/- 0.01, respectively. Further anisotropy decay after formation of the M intermediate in about 0.5 ms is evidence of orientational motion at this stage in the photocycle. A constant anisotropy with a value of 0.39 +/- 0.02 in the O intermediate demonstrates a recovery of the initial protein orientation with the formation of the O state. These results demonstrate that reorientations in BR are photoinduced and reversible. Similar measurements for L and M were carried out for purple membrane in polyacrylamide gels, where the anisotropies in the L and M states are 0.38 +/- 0.014 and 0.36 +/- 0.01, respectively. These results show that reorientations also occur in BR immobilized in gels. Anisotropy decay in the M state after formation of the M intermediate was not detected in the gels, in contrast to the M intermediate in suspensions. Orientational changes are observed for BR in purple membrane suspensions in the K state, during the K-->L step, in the M state possibly related to an M1-->M2 transition, and in the O state, where an almost complete return to the original orientation occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrasensitive time-resolved linear dichroism spectral measurements using near-crossed linear polarizers

A simple and sensitive technique is introduced to measure time-resolved linear dichroism of spectral transitions. This technique uses the fact that a linear dichroic sample rotates the polarization plane of linearly polarized light. The theoretical basis of the technique is presented using Mueller calculus and a detailed signal analysis is given to account for the effects of various optical imperfections. The results of this analysis are confirmed with the application of the technique to the time-resolved linear dichroism of bacteriorhodopsin in membrane patches (purple membrane) during its photocycle. These experimental results demonstrate the sentivity of the technique.