A statistical approach to the estimation of mechanical unfolding parameters from the unfolding patterns of protein heteropolymers.

A statistical calculation is described with which the saw-tooth-like unfolding patterns of concatenated heteropolymeric proteins can be used to estimate the forced unfolding parameters of a previously uncharacterized protein. The chance of observing the various sequences of unfolding events, such as ABAABBB or BBAAABB etc, for two proteins of types A and B is calculated using proteins with various ratios of A and B and at different values of effective unfolding rate constants. If the experimental rate constant for forced unfolding, k(0), and distance to the transition state x(u) are known for one protein, then the calculation allows an estimation of values for the other. The predictions are compared with Monte Carlo simulations and experimental data.

Picosecond optical correlation using dynamic holography in polyacetylene.

An image-processing system based on four-wave mixing in a film of polyacetylene less than 100 nm thick is demonstrated with a 1-ps processing cycle time. Image phase conjugation and cross correlation are performed with a resolution space-bandwidth product of 0.68 x 10(5) (equivalent to 261 x 261 pixels) in the phase-conjugate image. The light source was amplified optical pulses from a colliding-pulse mode-locked dye laser at a wavelength of 625 nm.

Studies of the fluorescence from tryptophan in melittin.

The fluorescence lifetime and rotational correlation time of the tryptophan residue in melittin, as both a monomer and tetramer, have been measured between pH 6 and 11. The fluorescence decays are non-exponential and give lifetimes of 0.7 +/- 0.1 ns and 3.1 +/- 0.1 ns. This emission is consistent with a model in which the tryptophan residue is in slightly different environments in the protein. In a dilute solution of monomer the mean fluorescence lifetime is 2.3 +/- 0.1 ns, below pH 10, but falls to 1.7 ns at higher pH. In contrast, the melittin tetramer has a mean fluorescence lifetime of only 2.2 ns at pH 6, which falls to 1.9 ns by pH 8, and falls again above pH 10 to the same value as in monomeric melittin. The behaviour between pH 6 and 8 is explained as the quenching of the Trp residue by lysine groups, which are near to the Trp in the tetramer but in the monomer, are too distant to quench. Fluorescence anisotropy decays show that the Trp residue has considerable freedom of motion and the range of "wobbling" motion is 35 +/- 10 degrees in the tetramer.

The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers.

The time-resolved fluorescence emission anisotropy of 12-(9-anthroyloxy)stearic acid (12-AS) and 1,6-diphenyl-1,3,5-hexatriene (DPH) have been measured in dipalmitoylphosphatidylcholine liposomes in the presence and absence of 40 mol% cholesterol at temperatures above and below the phase transition temperature (41 degrees C). By using a synchronously-pumped mode-locked frequency-doubled dye laser and single photon counting detection with an excitation response function of 300 picosecond, rotational correlation times down to less than 1 nanosecond could be resolved. Whereas DPH showed only small changes in the limiting anisotropy on the addition of cholesterol, 12-AS showed significant increases in this parameter with the effect being potentiated at higher temperatures. This difference in behaviour has been attributed to a fluorophore-cholesterol interaction that resulted in a change in the fluorophore geometry. Not only do DPH and 12-AS sense different depolarizing rotations due to the different directions of their emission dipoles but also differ in their lipid interactions which alter their limiting anisotropies. The implication is that the comparison of steady-state anisotropy measurements between chemically identical fluorophores in different lipid environments may be complicated by molecular distortions that change the motions to which the steady-state fluorescence parameters will be sensitive.

Secondary structure and dynamics of glucagon in solution.

A correlation between the secondary structure of glucagon determined by circular dichroism and its dynamic behaviour as obtained from picosecond fluorescence anisotropy is demonstrated. The CD data show that the percentage of alpha-helix decreases with increasing temperature, but the rotational relaxation time of the glucagon increases with temperature. These observations suggest that the protein's shape changes with temperature in such a way that its volume is larger at 38 degrees C than at 5.5 degrees C. The fluorescence anisotropy of glucagon decays biexponentially at each temperature studied and at 26 degrees C the rotation lifetimes are 1670 and 307 ps at pH 10.2 and 2147 and 517 ps at pH 2.2. It is proposed that the shorter decays are due to the restricted motion of the single tryptophan residue while rotation of the whole protein is responsible for the longer decays. The calculated rotational diffusion coefficient, Dw, of the tryptophan residue is much smaller, (ie. has a larger apparent volume) than that of a free tryptophan in solution. The hydrophobic interactions between residues Phe-22 to Leu-26 are probably responsible for the larger apparent volume in the protein compared to solution and will stabilize this part of the protein. The rotational diffusion of aggregated glucagon is also discussed.

Structural features in ethanol-water mixtures revealed by picosecond fluorescence anisotropy.

When an ensemble of molecules is excited with polarized light an anisotropic orientational distribution with respect to the transition dipole moment is produced. This anisotropy can decay in time due to the rotational motion of the molecules and consequently leads to depolarization of the fluorescence1-6. The rate of this rotational motion has been successfully predicted from hydrodynamic theory. How much the rotational relaxation depends on molecular geometry and how much on specific solvent-solute interactions has been studied by picosecond spectroscopy1-6 and other techniques7-9. In all cases so far reported, the rotational behaviour seems to be accounted for by the Debye-Stokes-Einstein (DSE) equation τR = f/kT. This relates the rotational relaxation time τR (inversely related to the rotational diffusion coefficient) to the frictional coefficient, f, which is proportional to the product of the shear viscosity, the molecular volume and a constant dependent on the 'stick' or 'slip' boundary conditions3,10,11. We report here, however, that large deviations from DSE behaviour have been observed in the rotational diffusion of the dye cresyl violet in ethanol-water mixtures. Different rotational relaxation times are observed in solutions of the same viscosity but differing composition. This behaviour can be rationalized using previously proposed models for water-ethanol mixtures.

Oligosaccharide motion in erythrocyte membranes investigated by picosecond fluorescence polarization and microsecond dichroism of an optical probe.

Oligosaccharide chains on the surfce of human erythrocytes were labeled with the probe eosin 5-thiosemicarbazide. The probe was conjugated to aldehydes produced by oxidation of sialic acid and galactose residues. The probe is associated mostly with glycophorin A after sialic acid labeling, whereas multiple components, including band 3 and lipids, are labeled after galactose oxidation. Fast molecular motion was studied by measuring steady-state and picosecond time-resolved fluorescence depolarization. Slower motions were investigated by observing flash-induced transient dichroism. It was found that both eosin-labeled sialic acid and galactose residues exhibit a rapid motion with correlation time of approximately 3 nsec. This motion is assigned to independent motion of the probe, possibly in conjunction with a short segment of the oligosaccharide chain. The order parameter of the fast motion is 0.8.-0.9, demonstrating that its angular amplitude is highly restricted. For eosin-labeled sialic acid, the order parameter in the microsecond time range is 0.2-0.3. It is deduced that a second, slower rotational motion is present, which is assigned to a cooperative motion of the oligosaccharide chains. The correlation time of this motion is in the range 10(-7)-(10-5) sec. Some eosin-labeled galactose residues may have a similar slow motion, but most appear to be remarkably immobile over the time range 10(-8)-10(-3) sec.

The fluorescence decay kinetics of in vivo chlorophyll measured using low intensity excitation.

We report fluorescence lifetimes for in vivo chlorophyll a using a time-correlated single-photon counting technique with tunable dye laser excitation. The fluorescence decay of dark-adapted chlorella is almost exponential with a lifetime of 490 ps, which is independent of excitation from 570 nm to 640 nm. Chloroplasts show a two-component decay of 410 ps and approximately 1.4 ns, the proportion of long component depending upon the fluorescence state of the chloroplasts. The fluorescence lifetime of Photosystem I was determined to be 110 ps from measurements on fragments enriched in Photosystem I prepared from chloroplasts with digitonin.

Energy transfer in a model of the photosynthetic unit.

A simple model of the photosynthetic unit has been constructed and used for simulated Förster-type energy migration, fluorescence and intersystem crossing, in order to gain insight into the conditions that influence both the form and the lifetime of the fluorescence decay in vivo. The model consists of a two-dimensional random lattice with one central trap. The simulation was done by means of repetitive Monte Carlo-type computations. The results obtained show that the form of the decay curve changes from exponential to non-exponential, as the chlorophyll concentration (molecules/nm2) is increased. The fluorescence lifetimes (tau 1/e) were also found to decrease substantially with only slight increases inc concentration. At a concentration comparable to that of chlorophyll in the chloroplast, both the form of the fluorescence decay and the lifetime are in fair agreement with experiment in vivo. The reasons for non-exponentially of the decay as well as the properties of energy migration are discussed. Preliminary work involving the dependence of trapping rate on donor concentration is also presented.

Excited state annihilation in the photosynthetic unit.

The kinetics of the in vivo fluorescence decays and fluorescence yields, as a function of excitation intensity, have been analysed with a model using excited state annihilation and time-dependent quenching processes. Triplet states, formed in the singlet-singlet annihilation processes, account for additional quenching of singlet states and the persistence of annihilation at longer times than the fluorescence life-time. Together these processes give a satisfactory account of existing experimental data of the intensity dependence of fluorescence in vivo.