Time-resolved Anisotropy Measurements Research Articles

An equilibrium study of the dependence of secondary and tertiary structure of creatine kinase on subunit association

Several physical properties of dimeric creatine kinase in increasing concentrations of guanidine hydrochloride (GDN/HC1) were evaluated and correlated with degree of subunit dissociation, determined by isozyme competitive hybridization. Three distinct stages were observed that correlated with phases before, during and after dissociation. In 0.2 M GDN/HC1, before significant dimer dissociation, creatine kinase has 75% of its original activity, and exhibits only small decreases in circular dichroism, intrinsic fluorescence and emission maximum. The spectral characteristics of creatine kinase (CK) derivatized with 5-[(((2-iodoacetyl)amino)ethyl)amino]naphthalene-l-sulfonate (AEDANS-CK) at the cysteine near the active site are suggestive of a slightly more non-polar environment. A decrease in steady-state anisotropy (0.140 to 0.132) was characterized by time-resolved methods. The slow component of the time-resolved anisotropy decay law, which reflects global protein rotation, is decreased only from 36.6 to 33.4 ns. The faster component decreases from 1.95 to 0.74 ns which suggests the active-site domain is more sensitive to conformation perturbation than the protein as a whole. Overall these observations suggest the subunits within the dimeric state are rather stable in dilute denaturant, but undergo a minor contraction in conformation. The region of the active site, as reported by the extrinsic fluorophore, is less polar but apparently more flexible in dilute denaturant. Between 0.5 M and 1 M GDN/HC1, most of the dimers dissociate, 63% of helical content is lost and inactivation is complete. The intrinsic fluorescence shifts 8 nm to the red and increases by 35%, indicating exposure of tryptophans to solvent and release of quenching, perhaps between residues on separate subunits. Over the same denaturant range, the spectral characteristics and lifetime of AEDANS-CK suggests less exposure of the active site to solvent. Time-resolved anisotropy measurements show that the sharp decrease in steady-state anisotropy to 0.086 is due to a decrease in macromolecular rotation to 22 ns. This may represent the rotational correlation time of a relatively intact subunit, and suggests limited subunit unfolding accompanying dissociation. Dissociation is complete in 1.5 M GDN/HC1. The subunits still retain 20% helical content in 2 M denaturant and not until 5 M GDN/HC1 is all helical structure eliminated. Above 2 M GDN/HCI, AEDANS-CK exhibits sharp decreases in steady-state anisotropy, fluorescence lifetime and the long-lived component in the time-resolved anisotropy decay law. These results reveal a catastrophic loss of tertiary structure by the subunits and may define the physical properties of the random coil.

Chlorosomes of green sulfur bacteria: Pigment composition and energy transfer

The pigment composition and energy transfer pathways in isolated chlorosomes ofChlorobium phaeovibrioides andChlorobium vibrioforme were studied by means of high performance liquid chromatography (HPLC) and picosecond absorbance difference spectroscopy. Analysis of pigment extracts of the chlorosomes revealed that they contain small amounts of bacteriochlorophyll (BChl)a esterified with phytol, whereas the BChlsc, d ande are predominantly esterified with farnesol. The chlorosomal BChla content inC. phaeovibrioides andC. vibrioforme was found to be 1.5% and 0.9%, respectively. The time resolved absorbance difference spectra showed a bleaching shifted to longer wavelengths as compared to the Qy absorption maxima and in chlorosomes ofC. vibrioforme also an absorbance increase at shorter wavelengths was observed. These spectral features were ascribed to excitation of oligomers of BChle and BChlc/d, respectively. 'One-color' and 'two-color' pump-probe kinetics ofC. phaeovibrioides showed rapid energy transfer to long-wavelength absorbing BChle oligomers, followed by trapping of excitations by BChla with a time constant of about 60 ps. Time resolved anisotropy measurements inC. vibrioforme showed randomization of excitations among BChla molecules with a time constant of about 20 ps, indicating that BChla in the baseplate is organized in clusters. One-color and two-color pump-probe measurements inC. vibrioforme showed rapid energy transfer from short-wavelength to long-wavelength absorbing oligomers with a time constant of about 11 ps. Trapping of excitations by BChla in this species could not be resolved unambiguously due to annihilation processes in the BChla clusters, but may occur with time constants of 15, 70 and 200 ps.

Luminescence studies of the conformational behaviour of hydrophobically-modified, water-soluble polymers

A range of hydrophobically-modified water-soluble polymers, based upon acrylic acid and methacrylic acid as hydrophilic components, has been studied using various luminescence techniques. Time-resolved anisotropy measurements, on fluorescently labelled macromolecules, have been used to quantify the extent to which the conformational behaviour of a given polyelectrolyte affects its segmental mobility, and to gauge how such behaviour can be controlled by hydrophobic modification. Room-temperature stabilized phosphorescence from the labels and/or occluded guests and energy transfer experiments have been used to investigate the nature of the hydrophobic domains formed in these polymers. Hydrophobic modification of polyelectrolytes can be used to enhance the scope of water-soluble macromolecules as solubilizing media for photochemical and spectroscopic applications.

Energy transfer and trapping of excitations in membranes of Heliobacterium chlorum at 15 K

The transfer of excitation energy in membrane fragments of Heliobacterium chlorum was studied at 15 K by picosecond transient absorption spectroscopy. Due to the high repetition rate of the laser pulses, the primary electron donor was in the oxidized state in essentially all reaction centers. The absorbance difference spectrum showed that excitations in the antenna rapidly accumulate on the long wavelength absorbing component bacteriochlorophyll (BChl) g 808. Analysis of the kinetics of excited antenna BChl g showed a lifetime of less than 1 ps for excitations on BChl g 778, whereas excited BChl g 793 showed a somewhat slower decay of about 2 ps. Time resolved anisotropy measurements showed a very rapid randomization of excitations amongst pigments with similar absorption spectrum within less than 1 ps. Kinetics as well as the absorbance difference spectrum showed that the decay of excitations accumulated on BChl g 808 was strongly inhomogeneous. About 50% of the excitations disappeared with a time constant of 4 ± 2 ps, while the others showed decays with time constants of 20 ± 5 ps and 100 ± 20 ps. The amplitude of the 20 ps component decreased with the energy density of the excitation pulses and this decay component is attributed, at least in part, to singlet-triplet quenching. The main relaxation process of excitations with a time constant of about 4 ps is most probably due to energy transfer to the oxidized reaction center. The 100 ps decay component is associated with BChls g 808 that are relatively isolated, either spatially or energetically.

Fluorescence depletion measurements in various experimental geometries provide true emission and absorption anisotropies for the study of protein rotation

Use of fluorescence depletion methods for measuring slow protein rotational diffusion has been limited by failure to obtain, from depletion data, well-defined anisotropy functions dependent on the distribution of either fluorophore emission or absorption transition dipoles, but not both. Such anisotropies would be directly comparable to those obtained from phosphorescence emission or triplet absorption measurements. We now describe such procedures applicable to cuvet and microscope experimental geometries, together with supporting experimental results. In cuvet measurements, the pump and probe beams are colinear and fluorescence is collected at 90° to this axis. The data analysis procedure for this geometry has been suggested by Wegener ( Biophys. J., 46 (1984) 795) and permits calculation of the absorption and emission anisotropies and the interdipole angle. In microscope experiments, fluorescence emission is collected along the pump/probe beam axis. For microscope measurements, a new experimental procedure permits evaluation of absorption and emission anisotropies when the interdipole angle is independently known. In either case multiple depletion measurements are required, each with different relative orientations of the probe beam polarization, pump beam polarization and emission polarizer axis. We have used these methods to calculate the time-dependent anisotropies for eosin-derivatized BSA rotation in glycerol solutions in both experimental geometries. These data correspond well with those obtained from time-resolved phosphorescence anisotropy measurements.

Auto-reconvolution: a new method of data retrieval from time-resolved emission anisotropy measurements

A new method, that of “auto-reconvolution”, for the recovery of rotational relaxation data from time-resolved anisotropy measurements (TRAMS) is reported. In this approach the observed time-dependent “parallel” component of luminescence intensity, I |( t), is used to deconvolute its orthogonal counterpart, I ⊥( t). The procedure has been validated in TRAMS experiments upon four fluorescently labelled polymers in dilute solution, following vertically polarized excitation using synchrotron radiation. In each case, excellent agreement with data retrieved by the impulse reconvolution method was achieved.

Rotational mobility of high-affinity epidermal growth factor receptors on the surface of living A431 cells.

The rotational diffusion of epidermal growth factor (EGF) bound to its specific receptor on the surface of human carcinoma A431 cells was studied by means of time-resolved phosphorescence anisotropy measurements. The rotational mobility was measured on the total population of EGF receptors by using a saturating concentration of EGF conjugated with a phosphorescent label, erythrosin, or on the subpopulation of high-affinity EGF receptors by using a low concentration of labeled EGF. At 4 degrees C, the rotational correlation times for both the high-affinity and total (mostly low affinity) receptor populations were in the range of 60-100 microns. Elevation of the temperature to 37 degrees C resulted in a lengthening of the rotational correlation time of the total receptor population to 200-300 microns, confirming a previous study of receptor microaggregation. The high-affinity EGF receptors were completely immobilized at 37 degrees C (rotational correlation time greater than 500 microns). The data are consistent with a model involving association of the cytoskeleton with the high-affinity receptors at 37 degrees C, but not at 4 degrees C.

Cholesterol dynamics in membranes

Time-resolved fluorescence anisotropy of the sterol analogue, cholestatrienol, and 13C nuclear magnetic resonance (NMR) spin lattice relaxation time (T1c) measurements of [13C4] labeled cholesterol were exploited to determine the correlation times characterizing the major modes of motion of cholesterol in unsonicated phospholipid multilamellar liposomes. Two modes of motion were found to be important: (a) rotational diffusion and (b) time dependence of the orientation of the director for axial diffusion, or "wobble." From the time-resolved fluorescence anisotropy decays of cholestatrienol in egg phosphatidylcholine (PC) bilayers, a value for tau perpendicular, the correlation time for wobble, of 0.9 x 10(-9) s and a value for S perpendicular, the order parameter characterizing the same motion, of 0.45 s were calculated. Both tau perpendicular and S perpendicular were relatively insensitive to temperature and cholesterol content of the membranes. The T1c measurements of [13C4] labeled cholesterol did not provide a quantitative determination of tau parallel, the correlation time for axial diffusion. T1c from the lipid hydrocarbon chains suggested a value for tau perpendicular similar to that for cholesterol. Steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in a variety of pure and mixed lipid multilamellar liposomes. Both the lipid headgroups and the lipid hydrocarbons chains contributed to the determination of the sterol environment in the membrane, as revealed by these fluorescence measurements. In particular, effects of the phosphatidylethanolamine (PE) headgroup and of multiple unsaturation in the lipid hydrocarbon chains were observed. However, while the steady-state anisotropy was sensitive to these factors, the time-resolved fluorescence analysis indicated that tau perpendicular was not strongly affected by the lipid composition of the membrane. S perpendicular may be increased by the presence of PE. Both steady-state anisotropy measurements and time-resolved anisotropy measurements of cholestatrienol were used to probe sterol behavior in three biological membranes: bovine rod outer segment (ROS) disk membranes, human erythrocyte plasma membranes, and light rabbit muscle sarcoplasmic reticulum membranes. In the ROS disk membranes the value for S perpendicular was marginally higher than in the PC membranes, perhaps reflecting the influence of PE. The dramatic difference noted was in the value for tau perpendicular. In both the ROS disk membranes and the erythrocyte membranes, tau perpendicular was one-third to one-fifth of tau perpendicular in the phospholipid bilayers. This result may reveal an influence of membrane proteins on sterol behavior.

Sugar transport by the bacterial phosphotransferase system. Fluorescence studies of subunit interactions of enzyme I.

Enzyme I of the bacterial phosphoenolpyruvate:glycose phosphotransferase system (PTS) exhibits a temperature-dependent monomer/dimer equilibrium. The accompanying paper (Han, M. K., Roseman, S., and Brand, L. (1990) J. Biol. Chem. 265, 1985-1995) shows that the C-terminal -SH residue (Cys-575) can be modified specifically with fluorescent probes such as pyrene maleimide. The derivative retains full enzyme activity, and is capable of forming dimers at room temperature. In the present studies, Enzyme I labeled in this way is found to exhibit a temperature-, concentration-, and pH-dependent monomer/dimer association. The kinetics of dimer formation of Enzyme I is measured in the following way. A derivatized Enzyme I sample is prepared with a pyrene moiety irreversibly attached to the C-terminal -SH residue and 5,5'-dithiobis-2-nitrobenzoic acid reversibly attached to the other 3 -SH residues. This modified enzyme does not form dimers at room temperature. Addition of dithiothreitol results in total release of the thionitrobenzoate anion within 2 min. After the three -SH groups are unblocked, steady-state and nanosecond time-resolved emission anisotropy measurements indicate the dimer is formed over a period of 30 min. In a similar experiment, little dimer formation is observed at 3 degrees C, at temperature at which the native enzyme also does not form dimers. Tryptophan fluorescence is also examined during the release of the thionitrobenzoate. After the completion of thionitrobenzoate release, additional slow steady-state tryptophan fluorescence changes are observed. These results suggest that dimer formation may be preceded by a conformational change following thionitrobenzoate release.

Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome- b5 reductase systems in phosphatidylcholine vesicles

The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeld cytochrome b 5 (DNS-cytochrome b 5), on interaction with NADH-cytochrome- b 5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b 5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b 5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b 5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15–30°C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b 5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15–30°C. The enzyme activity of the DNS-cytochrome- b 5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b 5 and NADH-cytochrome- b 5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.

The membrane fluidity concept revisited by polarized fluorescence spectroscopy on different model membranes containing unsaturated lipids and sterols

Quantitative analysis of time-resolved anisotropy measurements of DPH or TMA-DPH in lipid vesicles yields more than one mathematically correct solution. The solutions differ with respect to the average orientation and to the reorientational dynamics of the probe molecules in the bilayer. This leads to quite opposite results regarding the effects of cholesterol on membrane fluidity. One solution predicts an increase in fluidity, the other a decrease. Angle-resolved fluorescence depolarization (AFD) measurements of probes in oriented lipid bilayers enable determination of the average orientation of the probes in the bilayer and, if the fluorescence decay function is known, of the reorientational dynamics. Analysis of AFD measurements of DPH and TMA-DPH show that increasing unsaturation leads to a decrease in molecular order and a decrease in reorientational dynamics (= fluidit) of the probes. At temperatures above the phase transition of the lipids, the addition of cholesterol causes an increase in molecular order and an increase in reorientational dynamics (= fluidty). The plant sterol stigmasterol, which is structurally closely related to cholesterol, has different effects than cholesterol. The effects vary with the structure of the surrounding lipids. The membrane fluidity concept as it was originally proposed by Chapman [29, 30] attempts to describe the structural and dynamic properties of lipids in a membrane using one single parameter indicated as ‘membrane fluidity’. Our results show that it is necessary to distinguish between structural parameters describing molecular order and motion parameters describing molecular dynamics, thus supporting a similar suggestion by Seelig and Seelig [19, 21]. In order to be useful, the membrane fluidity concept has to be limited to the parameters describing molecular dynamics.

A time-resolved fluorescence anisotropy study of bilayer membranes containing α-tocopherol

Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing alpha-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing alpha-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly.

A multiplexed single-photon instrument for routine measurement of time-resolved fluorescence anisotropy

The authors report a new type of instrument, the Edinburgh Instruments Model 2997 differential fluorometer, for the measurement of the decay of fluorescence anisotropy. The instrument incorporates three multiplexed detection channels for simultaneous measurement of two polarised fluorescence decays and the excitation pulse, using time-correlated single-photon counting. This arrangement corrects automatically for temporal and intensity fluctuations of the spark source. A study of the probe 1,6-diphenyl-1,3,5-hexatriene (DPH) in a white oil reveals that the instrument can measure rotational correlation times with subnanosecond time resolution in a third of the time of conventional methods.

Rotational dynamics of the Fc receptor for immunoglobulin E on histamine-releasing rat basophilic leukemia cells

The rotational diffusion of immunoglobulin E (IgE) bound to its specific Fc receptor on the surface of living rat basophilic leukemia cells was determined from time-resolved phosphorescence emission and anisotropy measurements. The IgE-receptor complexes are mobile throughout the range of temperatures of 5-38 degrees C. The residual anisotropy does not reach zero, indicating that the rotational diffusion is hindered. The values of rotational correlation times for each temperature are consistent with dispersed receptors rotating freely in the cell membrane and rule out any significant aggregation of occupied receptors before cross-linking by antigen or anti-IgE antibodies. The rotational correlation times decrease with increasing temperature from 65 microseconds at 5.5 degrees C to 23 microseconds at 38 degrees C. However, the degree of orientational constraint experienced by the probe is unchanged. Thus, the temperature dependence can be attributed primarily to a change in the effective viscosity of the cellular plasma membrane. The phosphorescence depolarization technique is very sensitive (our probe concentrations were 10-100 nM) and thus generally applicable to studies of surface receptors and antigens on living cells.

Pulse fluorometry using simultaneous acquisition of fluorescence and excitation

We report a new method of measuring fluorescence lifetimes which uses the single-photon technique and two detection channels with matched impulse response for simultaneous acquisition of fluorescence and excitation (SAFE). This differential arrangement is shown to correct automatically for variations in the optical pulse profile during the measurement, thus eliminating a common source of error. It can be used to improve precision and sensitivity with any pulsed light source such as a flashlamp, laser, or synchrotron. A routing system separates photomultiplier coincidences from the dual detection channels into different memory segments of a multichannel analyzer (MCA) using a single time-to-amplitude converter (TAC). Comprehensive data are presented on tuning the single-photon response of the Philips range of XP2020Q photomultipliers. Results obtained using a coaxial flashlamp to excite a dilute solution of PPO in ethanol give a lifetime of 1.63±0.02 ns in good agreement with that obtained using conventional fluorometry. The method is also useful in the study of dual emissions such as in monomer–excimer systems and in the measurement of time-resolved emission anisotropy.

Distribution and mobility of murine histocompatibility H-2Kk antigen in the cytoplasmic membrane.

The topographical distributions and mobilities of the murine histocompatibility antigen H-2Kk and of concanavalin A (Con A) binding sites have been studied on a murine lymphoma cell line. The spatial distribution of H-2Kk antigens, the average distance between H-2Kk antigens and Con A binding sites, and the separation of different determinants on the H-2Kk antigen itself were determined by using fluorescence resonance energy-transfer measurements with a dual-laser flow sorter. From the lack of energy transfer between bound monoclonal anti-H-2Kk antibodies conjugated with fluorescein (donor) and rhodamine (acceptor), we conclude that the H-2Kk antigen exists without appreciable clustering on the cell surface. Substantial energy transfer between appropriately labeled Con A and antibodies bound to the H-2Kk antigen shows that the two populations are interspersed. Donor/acceptor pairs of monoclonal antibodies binding to different determinants on the same H-2Kk antigen exhibited a degree of energy transfer indicative of a mean separation of 8.6 nm between the sites. Time-resolved phosphorescence anisotropy measurements with anti-H-2Kk antibodies labeled with eosin or erythrosin yielded rotational mobility information for the antigen-antibody complexes on the cell membrane. The rotational correlation time of 10-20 mus and the finite residual anisotropy are compatible with an uniaxial mode of rotation of monomeric antigen around its transmembrane portion and, thus, provide additional evidence for an unclustered distribution. Capping by rabbit anti-mouse IgG immobilized the antigen-antibody complex. Fluorescence recovery after photobleaching was used to calculate an apparent lateral diffusion coefficient of 5 +/- 3 X 10(-10) cm2 . s-1 for the H-2Kk antigen labeled with fluoresceinated IgG or its corresponding Fab fragment.