Conformation and orientation of the retinyl chromophore in rhodopsin: a critical evaluation of recent NMR data on the basis of theoretical calculations results in a minimum energy structure consistent with all experimental data.

In the absence of a high-resolution diffraction structure, the orientation and conformation of the protonated Schiffs base retinylidinium chromophore of rhodopsin within the opsin matrix has been the subject of much speculation. There have been two recent reliable and precise NMR results that bear on this issue. One involves a determination of the C20-C10 and C20-C11 distances by Verdegem et al. [Biochemistry 38, 11316-11324 (1999)]. The other is the determination of the orientation of the methine C to methyl group vectors C5-C18, C9-C19, and C13-C20 relative to the membrane normal by Gröbner et al. [Nature 405 (6788), 810-813 (2000)]. Using molecular orbital methods that include extensive configuration interaction, we have determined what we propose to be the minimum energy conformation of this chromophore. The above NMR results permit us to check this structure in the C10-C11=C12-C13 region and then to check the global structure via the relative orientation of the three C18, C19, and C20 methyl groups. This method provides a detailed structure and also the orientation for the retinyl chromophore relative to the membrane normal and argues strongly that the protein does not appreciably alter the chromophore geometry from its minimum energy configuration that is nearly planar s-trans at the 6-7 bond. Finally, the chromophore structure and orientation presented in the recently published X-ray diffraction structure is compared with our proposed structure and with the deuterium NMR results.

Histidine-tryptophan interactions in T4 lysozyme: 'anomalous' pH dependence of fluorescence.

A variant of T4 lysozyme which contains only a single tryptophan residue (at position 138) has been prepared (W126Y/W158Y designated 'YWY'). Two additional mutations to YWY have been prepared involving replacement of glutamine 105, which hydrogen bonds to the indole N-H of trp 138 in wild type, with either a histidine (YWY/Q105H) or an alanine (YWY/Q105A). The fluorescence properties of these two species are investigated as a function of pH. YWY/Q105A exhibits essentially a single exponential fluorescence decay (5% tau = 0.35 ns 95% tau = 5 ns) and almost no pH dependence in steady state or time resolved fluorescence behavior. In contrast, YWY/Q105H exhibits complex fluorescence decay over the entire pH range used in these experiments. As the pH is lowered from 8 to 4, there is an increase in the quantum yield and a change in the average lifetime (from 2.0 to 3.1 ns). Using this data, the pKa of histidine 105 has been determined to be 5.9. These results are contrasted to those from other proteins which show a pH dependent tryptophan fluorescence associated with a neighboring histidine or other residue. Quenching behavior in terms of the stereochemistry of the tryptophan-histidine interaction and implications of these results for current models of complex fluorescence behavior of single tryptophan proteins are also discussed.

Photophysics of tryptophan in bacteriophage T4 lysozymes.

Bacteriophage T4 lysozyme contains three tryptophan residues in distinct environments. Lysozymes with one or two of these residues replaced by tyrosine are used to characterize the photophysics of tryptophan in these individual sites. The fluorescence spectra, average lifetimes, and quantum yields of these three single-tryptophan variants are understandable in terms of the neighboring residues. The emission spectra and radiative lifetimes are found to be the same for all three species while the quantum yield and decay kinetics are quite distinct. The variation of the average nonradiative rate constant is correlated with neighboring quenching groups. Quenching by I- correlates with exposure of the tryptophan residue based on the crystal structure. Complex behavior is observed for the time dependence of the fluorescence decay in all three cases, including that of the immobile tryptophan-138 residue. The complexity of the fluorescence decay is ascribed to heterogeneity in the nonradiative rate constant among microstates. Energy transfer between tryptophan residues is inferred to occur from comparison of the quantum yields of the two-tryptophan and single-tryptophan proteins and is discussed in terms of the Förster mechanism.

Environmental modulation of M13 coat protein tryptophan fluorescence dynamics.

The effects of detergent [deoxycholate (DOC) and phospholipid [dimyristoylphosphatidylcholine (DMPC)] environments on the rotational dynamics of the single tryptophan residue 26 of bacteriophage M13 coat protein have been investigated by using time-resolved single photon counting measurements of the fluorescence intensity and anisotropy decay. The total fluorescence decay of tryptophan-26 is complex but rather similar in DOC as compared to DMPC when analyzed in terms of a lifetime distribution (exponential series method). This similarity, in conjunction with the almost identical steady-state fluorescence spectra, indicates only minor differences between the tryptophan environments in DOC and DMPC. The reorientational dynamics of tryptophan-26 are dominated by slow rotation of the entire protein in both detergent and phospholipid environments. The resolved anisotropy decay in DOC can be approximated by a simple hydrodynamic model of protein/detergent micelle rotational diffusion, although the data indicative slightly greater complexity in the rotational motion. The tryptophan fluorescence anisotropy is not sensitive to protein conformational changes in DOC detected by nuclear magnetic resonance on the basis of pH independence in the range 7.5-9.1. In DMPC bilayers, restricted tryptophan motion with a correlation time of approximately 2 ns is observed together with a second very slow reorientational component. Resolution of the time constant for this slow rotation is obscured by the tryptophan fluorescence time window being too short to clearly locate its anisotropic limit. The possible contribution made by axial rotational diffusion of the protein to this slow rotational process is discussed.(ABSTRACT TRUNCATED AT 250 WORDS)

Rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex. A steady-state and time-resolved anisotropy study.

The rotational dynamics of the single tryptophan of porcine pancreatic phospholipase A2 and its zymogen (prophospholipase A2) have been studied by polarized fluorescence using steady-state and time-resolved single-photon counting techniques. The motion of Trp-3 in phospholipase A2 consists of a rapid subnanosecond wobble of the indole ring with an amplitude of about +/- 20 degrees accompanied by slower isotropic rotation of the entire protein. The rotational correlation times for overall particle rotational diffusion are consistent with conventional hydrodynamic theory. When phospholipase A2 binds to micelles of n-hexadecylphosphocholine, the amplitude of the fast ring rotation decreases. The whole particle rotational correlation time of the enzyme/micelle complex is smaller than the minimum value calculated from hydrodynamic theory. A similar result is obtained for the micelle itself by using the lipophilic probe transparinaric acid. These low values for the particle correlation times can be understood by postulating that an isotropic motion of the fluorophore in the small detergent particles contributes to the angular reorientation of the fluorophore. The internal reorientational motion of the tryptophan in the zymogen, prophospholipase A2, is of larger amplitude than that observed for the enzyme; specifically, the proenzyme exhibits a motion with a significant amplitude on the nanosecond time scale. This additional freedom of motion is attributed to segmental mobility of the N-terminal residues of prophospholipase A2. This demonstrates that this region of the protein is flexible in the zymogen but not in the processed enzyme. The implications of these findings for the mechanism of surface activation of phospholipase A2 are discussed by analogy with a trypsinogen-trypsin activation model.

Complex photophysics of the single tryptophan of porcine pancreatic phospholipase A2, its zymogen, and an enzyme/micelle complex.

The fluorescence emission of the single tryptophan in porcine pancreatic phospholipase A2, its zymogen, and a micellar complex of the enzyme with the nonhydrolyzable substrate analogue n-hexadecylphosphocholine has been studied by both steady-state and time-resolved techniques. Stern-Volmer quenching studies with acrylamide indicate that, both in the enzyme and in the zymogen, the tryptophan is exposed to solvent. Similar studies with ionic quenchers show that there is appreciable ionic character to the tryptophan environment. Single photon counting fluorescence measurements were performed using a high repetition rate synchronously pumped dye laser as a light source. When tryptophan fluorescence is collected with a broad-band (80-nm) emission filter, the decay kinetics in the enzyme and the zymogen require at least three, and often four, exponential terms for a proper description. The decay kinetics can be adequately described by three exponential terms when the fluorescence is collected at specific wavelengths by using narrow (10-nm) band-pass filters. The lifetimes are approximately constant across the emission band, but the amplitudes vary with the fraction of the long lifetime increasing at longer emission wavelengths. Formation of a complex between phospholipase A2 and micelles of n-hexadecylphospohocholine produces large changes in the tryptophan emission that are associated with transfer to a hydrophobic environment. The decay kinetics of tryptophan in the enzyme/micelle complex appears to require only two exponential terms. This is the first reported instance of fluorescence data from a single tryptophan protein requiring more than double-exponential decay kinetics. The results are discussed in terms of the range of environments sampled by the tryptophan residue and the resulting distribution of lifetimes.

Bilayer acyl chain dynamics and lipid-protein interaction: the effect of the M13 bacteriophage coat protein on the decay of the fluorescence anisotropy of parinaric acid.

Nanosecond fluorescence polarization anisotropy decay is used to determine the effect of the bacteriophage M13 coat protein on lipid bilayer acyl chain dynamics and order. The fluorescent acyl chain analogues cis- and trans-parinaric acid were used to determine the rate and extent of the angular motion of acyl chains in liquid crystalline (39 degrees C) dimyristoylphosphatidylcholine bilayers free of coat protein or containing the coat protein at a protein:lipid ratio of 1:30. Subnanosecond time resolution was obtained by using synchrotron radiation as the excitation source for single photon counting detection. Previous measurements of Förster energy transfer from coat protein tryptophan to cis- or trans-parinaric acid have shown that these probes are randomly distributed in the bilayer with respect to the protein. The anisotropy decay observed for pure bilayers has the form of a rapid drop, followed by a nonzero constant region extending from roughly 3 ns to at least 12 ns. The magnitude of the anisotropy in the plateau region is simply related to the acyl chain order parameter. The effect of the M13 coat protein is to increase the acyl chain order parameter significantly while having only a small effect on the rate of angular relaxation. This behavior is rationalized in terms of a simple microscopic model. The order parameters for pure lipid and coat protein containing bilayers are compared to 2H-NMR values.

Fluorescence lifetime and time-resolved polarization anisotropy studies of acyl chain order and dynamics in lipid bilayers.

The time-resolved fluorescence intensity and anisotropy decays of cis- and trans-parinaric acids and phosphatidylcholines labeled with trans-parinaric acid have been characterized in bilayers formed by several phosphatidylcholines and by dipalmitoylphosphatidylcholine-cholesterol mixtures, at several temperatures. Both a conventional free-running nitrogen flashlamp and the novel synchrotron source at the Stanford Linear Accelerator Center (SLAC) were used as excitation sources for a modified single photon counting fluorescence lifetime apparatus. The measured emission decay kinetics of both isomers of parinaric acid were biexponential in all but one of the lipid systems examined. The fluorescence anisotropy of parinaric acid was large and constant in gel phase lipids, but showed a very rapid (approximately 2 GHz) decay of large amplitude in fluid lipids. In all lipid systems studied, the fluorescence anisotropy decayed to a nonzero asymptote, in striking contrast to the behavior observed in viscous solvent solutions. The asymptotic anisotropy was used to calculate an "order parameter" of the emission transition dipole. The value of the order parameter is quite close to that obtained by deuterium NMR. Cholesterol increased the order parameter measured in fluid dipalmitoylphosphatidylcholine but did not substantially affect the rate of angular relaxation. Experiments conducted with trans-parinaroylphosphatidylcholines yielded results virtually identical with those obtained with trans-parinaric acid.

A theory of the effects of head-group structure and chain unsaturation on the chain melting transition of phospholipid dispersions.

We have developed statistical mechanical descriptions of the effects of head-group structure and acyl chain unsaturation on the chain melting phase transition of aqueous dispersions of bilayers containing glycerophosphocholines and glycerophosphoethanolamines. The theoretical framework is an extension of the model of Jacobs et al. [Jacobs, R. E., Hudson, B. S., & Andersen, H. C. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 3993]. There are several systematic trends in the experimental transition data for various types of phospholipids. Assumptions about the physical origins of these trends were incorporated into statistical mechanical models, which were used to calculate transition temperatures and enthalpies. The extent to which the calculated results of a model reproduce the experimental trends is taken as a measure of the validity of the assumptions on which the model is based. We found that the gross differences among the transition temperatures of phospholipids with two saturated chains, two trans-unsaturated chains, two cis-unsaturated chains, and one cis-unsaturated and one saturated chain can all be explained in terms of the effect of the double bonds on molecular shape and the subsequent effect of shape on the ability of molecules to pack together into a low-energy state at high density. The dependence of transition temperature on the location of the double bond in cis-unsaturated molecules can be understood on the same basis. The differences between the transition temperatures of glycerophosphocholines and glycerophosphoethanolamines with the same hydrocarbon chains can be explained in terms of a larger intermolecular attraction (or smaller repulsion) for the latter than for the former. These differences depend on the presence or absence of unsaturation in the hydrocarbon chains in a way that is consistent with the postulate that hydrogen bonding between glycerophosphoethanolamines is responsible for the differences.

An analytic solution to the Förster energy transfer problem in two dimensions.

An analytic solution of the Förster energy transfer problem in two dimensions is presented for the case in which the orientation factor is independent of the donor-acceptor distance, and both the donors and acceptors are randomly distributed in a plane. A general solution based on the method of Förster is possible since all distances are measured in units of R0. The analytic solution is extended to the cases of donors embedded in structures that exclude acceptors, and donors that bind acceptors. The validity of the analytic solutions is demonstrated by comparison with numerical simulation calculations. Numerical approximations to the exact solutions are given for ease of computation. Specific applications to the case of fluorescence quenching of a membrane-bound donor by membrane-bound acceptors are presented.

Human serum albumin. Spectroscopic studies of binding and proximity relationships for fatty acids and bilirubin.

Binding and proximity relationships of hydrophobic ligands on human serum albumin have been studied using absorption, fluorescence, circular dichroism, and electron paramagnetic resonance spectroscopy. The ligands studied were bilirubin, two conjugated linear polyene fatty acids, cis-parinaric acid and cis-eleostearic acid, and three nitroxide derivatives of stearic acid with doxyl groups at positions 5, 10, and 12, respectively. Binding of polyene fatty acids was monitored by absorption peak shifts, induced circular dichroism, enhancement of fluorescence, and energy transfer between albumin's single tryptophanyl residue and the polyene chromophore. Induced circular dichroism studies indicate excitonic ligand-ligand interaction between bound fatty acids. Fluorescence enhancement of cis-parinaric acid was analyzed using a stepwise multiple equilibrium model, and six binding constants in the range 10(8) to 10(6) M-1 were obtained, in agreement with previous measurements for other fatty acids. The temperature dependence of the equilibrium constants indicates that the binding enthalpy is nearly zero. Fluorescence energy transfer was similarly used to quantitate bilirubin binding to albumin. Energy transfer, nitroxide quenching of fluorescence, and electron paramagnetic resonance spectroscopy were used to elucidate binding geometries which support and extend proposed structural models for albumin. It is suggested that the first two fatty acids bind side-by-side in an antiparallel fashion in domain III of human serum albumin.

A theory of phase transitions and phase diagrams for one- and two-component phospholipid bilayers.

A statistical mechanical partition function for phospholipid bilayers is constructed to obtain a theoretical description of the chain melting phase transition in lipid bilayer membranes and of the phase diagrams for two-component bilayers. In addition to providing an accurate representation of the transition temperatures and enthalpies of one-component bilayers composed of 1,2-diacylphosphatidylcholines, the theory can also account for the shapes of the phase diagrams observed for bilayers which are binary mixtures of these compounds with two different hydrocarbon chain lenghts.

Conjugated polyene fatty acids on fluorescent probes: spectroscopic characterization.

This paper is the first in a series which extends introductory studies of parinaric acid and its phospholipid derivatives as membrane probes (Sklar, L.A., Hudson, B., and Simoni, R.D. (1975), Proc. Natl. Acad. Sci. after U.S.A. 72, 1649; (1976), J. Supramol. Struct. 4, 449). Parinaric acid has a conjugated tetraene chromophore and exhibits many spectroscopic properties common to linear polyenes. Its absorption spectrum is characterized by a strong near-ultraviolet transition with vibronic structure, which is strongly affected by solvent polarizability. The fluorescence emission occurs at considerably lower energy than the absorption and the wavelength of the emission is nearly independent of the solvent. The fluorescence quantum yield and lifetime are strongly affected by temperature and solvent. These spectral features are interpreted in terms of an excited electronic-state order such that a weak transition occurs at longer wavelengths than the strongly allowed transition which dominates the absorption. The sensitivity of the fluorescence quantum yield an lifetime to environment is shown to be due primarily to variations in the nonradiative rate, although changes in the radiative rate constant are also observed and interpreted. The absorption spectrum (epsilon max greater than 65 000) is in the 300-320-nm range, a region relatively free of absorption due to intrinsic biological chromophores. Shifts of several nanometers are characteristic of different environments. These shifts are compared to similar effects observed for a series of diphenylpolyenes for which new data are given and are correlated using a simple but adequate theory of solvent shifts. The intrinsic (or radiative) fluorescence lifetime is near 100 ns in a wide variety of environments. This is much longer than the intrinsic lifetime calculated from the absorption spectrum and strongly supports the proposed excited-state order.

Conjugated polyene fatty acids as fluorescent probes: synthetic phospholipid membrane studies.

The preparation of polyene fatty acid membrane probes cis- and trans-parinaric acid and parinaroylphosphatidylcholines and their use in studies of several one- and two- component lipid systems are described. The fluorescence quantum yield of trans-parinaric acid in dipalmitoylphosphatidylcholine at 20 degrees C is approximately 0.3; the quantum yield in aqueous solution is negligibly small. Thermal-phase transitions in single-component phospholipid dispersions are monitored with absorption and fluorescence excitation peak position, fluorescence intensity, lifetime, and polarization. The transition temperatures observed are consistent with previous determinations. Shifts in the absorption peak position are related to the bilayer expansion as it undergoes the gel to liquid-crystalline transition, while fluorescence depolarization provides semiquantitative information concerning molecular motion of the probe in the bilayer. A long fluorescence lifetime component is observed for parinaric acid in the solid phase (up to 50 ns), and a short lifetime component is observed (ca. 5 ns) in the fluid phase of dipalmitoylphosphatidylcholine; both lifetime components are observed in the transition region. In most phospholipids, cis-parinaric acid detects the melting transition at about 1 degree C lower than trans-parinaric acid. Partitioning experiments involving mixed populations of phospholipid vesicles show that trans-parinaric acid preferentially associates with solid-phase lipids, while cis-parinaric acid shows a more equal distribution between solid and fluid lipids. The binding of cis-parinaric acid to dipalmitoylphosphatidylcholine at 25 degrees C is described as partitioning of parinaric acid between lipid vesicles and the aqueous phase with a partition coefficient of 5 X 10(5). Several rates are observed in the binding process which are interpreted as rapid outer monolayer uptake and a much slower process of interlamellar exchange. The phase diagram of the binary lipid mixture dipalmitoylphosphatidylcholine-dipalmitoylphosphatidylethanolamine has also been examined and found to be essentially identical to the one constructed using a nitroxide probe.

Conjugated polyene fatty acids as fluorescent probes: biosynthetic incorporation of parinaric acid by Escherichia coli and studies of phase transitions.

The use of the fluorescent fatty acid, parinaric acid (9, 11, 13, 15-octadecatetraenoic acid) (PnA), was studied in cells of an unsaturated fatty acid auxotroph of Escherichia coli. Growth conditions were found that permitted biosynthetic incorporation of PnA (up to 3%) into membrane phospholipids during growth on oleic or elaidic acid. Fluorescence measurements of incorporated PnA revealed phase transitions in cells, membranes, and phospholipids at temperatures that reflected the fatty acid composition of the sample. Transitions had a well-defined onset from high temperature, while the lower and end point was less well defined. cis- and trans-PnA (cis, trnas, trans, cis, and all trans, respectively) gave comparable results. Similar phase transitions were detected with PnA, which was not biosynthetically incorporated. Fluorescence of tryptophan was measured in E. coli membranes as a function of concentration of PnA. Significant quenching of tryptophan fluorescence by PnA was observed.