Membranes anisotropy

Bemert JT, Groce DF. 1984. Acute response of rat liver microsomal lipids, lipid peroxidation, and membrane anisotropy to a single oral dose of polybrominated biphenyls. J Toxicol Environ Health 13 673-687. 

Microindentation anisotropy 143, 145 Microspherulitic structure 139 Microvoiding 193 Microvoids 194, 205 Model membranes 49, 53, 55 Modulus 135 Moisture sensitivity 199... 

Despite its weakness, the anisotropy of the g tensor of iron-sulfur centers can be used to determine the orientation of these centers or that of the accommodating polypeptide in relation to a more complex system such as a membrane-bound complex. For this purpose, the EPR study has to be carried out on either partially or fully oriented systems (oriented membranes or monocrystals, respectively). Lastly, the sensitivity of the EPR spectra of iron-sulfur centers to structural changes can be utilized to monitor the conformational changes induced in the protein by different factors, such as the pH and the ionic strength of the solvent or the binding of substrates and inhibitors. We return to the latter point in Section IV. 

Socaciu, C., R. Jessel, and H.A. Diehl. 2000. Competitive carotenoid and cholesterol incorporation into liposomes Effects on membrane phase transition, fluidity, polarity and anisotropy. Chem. Phys. Lipids 106 79-88. 

The versatility of luminescence goes beyond intensity-, wavelength- and kinetic-based measurements. Fluorescence polarization (or anisotropy) is an additional parameter still largely unexplored for optical sensing yet widely used in Biochemistry to study the interaction of proteins, the microfluidity of cell membranes and in fluorescence immunoassays. Although only a few optosensors based on luminescence polarization measurements can be found in the literature, elegant devices have recently been reported to measure chemical parameters such as pFI or O2 even with the bare eye41. 

The second issue concerns the anisotropy of the membrane. The models presented in this section all assume that the membrane has the symmetry of a chiral smectic-C liquid crystal, so that the only anisotropy in the membrane plane comes from the direction of the molecular tilt. With this assumption, the membrane has a twofold rotational symmetry about an axis in the membrane plane, perpendicular to the tilt direction. It is possible that a membrane... 

It is also possible that a membrane might have an even lower symmetry than a chiral smectic-C liquid crystal in particular, it might lose the twofold rotational symmetry. This would occur if the molecular tilt defines one orientation in the membrane plane and the direction of one-dimensional chains defines another orientation. In that case, the free energy would take a form similar to Eq. (5) but with additional elastic constants favoring curvature. The argument for tubule formation presented above would still apply, but it would become more mathematically complex because of the extra elastic constants. As an approximation, we can suppose that there is one principal direction of elastic anisotropy, with some slight perturbations about the ideal twofold symmetry. In that approximation, we can use the results presented above, with 4) representing the orientation of the principal elastic anisotropy. 

Salamon, Z. Tollin, G., Graphical analysis of mass and anisotropy changes observed by plasmon waveguide resonance spectroscopy can provide useful insights into membrane pro tein function, Biophys. J. 2004, 86, 2508 2516... 

Zdzislaw, S. Gordon, T., Optical anisotropy in lipid bilayer membranes Coupled plasmon waveguide resonance measurements of molecular orientation, polarizability, and shape, Biophys. J. 2001, 80, 1557 1567... 

The surface tension defined above was related to an interface that behaved mechanically as a membrane stretched uniformly and isotropically by a force which is the same at all points on the surface. A surface property defined this way is not always applicable to the surfaces of solids and the surface energy of planar surfaces is defined to take anisotropy into account. The surface energy is often in the literature interchanged with surface tension without further notice. Although this may be useful in practice, it is strictly not correct. 

It is interesting to note that rc(l — r.y,/r0) is exactly the area A under [r(t) — r, /ro. Therefore, even if the anisotropy decay is not a single exponential, Dw can be determined by means of Eq. (5.50) in which tc(1 — roo/ro) is replaced by the measured area A. An example of application of the wobble-in-cone model to the study of vesicles and membranes is given in Chapter 8 (Box 8.3). More general theories have also been developed (see Box 5.4). 

Lipari G. and Szabo A. (1980) Effect of Vibrational Motion on Fluorescence Depolarization and Nuclear Magnetic Resonance Relaxation in Macromolecules and Membranes, Biophys. J. 30, 489—506. Steiner R. F. (1991) Fluorescence Anisotropy Theory and Applications, in Lakowicz J. R. (Ed.), Topics in Fluorescence Spectroscopy, Vol. 2, Principles, Plenum Press, New York, pp. 127-176. 

Time-resolved emission anisotropy experiments provide information not only on the fluidity via the correlation time rc, but also on the order of the medium via the ratio rco/ro. The theoretical aspects are presented in Section 5.5.2, with special attention to the wobble-in-cone model (Kinosita et al., 1977 Lipari and Szabo, 1980). Phospholipid vesicles and natural membranes have been extensively studied by time-resolved fluorescence anisotropy. An illustration is given in Box 8.3. 

Lipid-protein interactions are of major importance in the structural and dynamic properties of biological membranes. Fluorescent probes can provide much information on these interactions. For example, van Paridon et al.a) used a synthetic derivative of phosphatidylinositol (PI) with a ris-parinaric acid (see formula in Figure 8.4) covalently linked on the sn-2 position for probing phospholipid vesicles and biological membranes. The emission anisotropy decays of this 2-parinaroyl-phosphatidylinositol (PPI) probe incorporated into vesicles consisting of phosphatidylcholine (PC) (with a fraction of 5 mol % of PI) and into acetylcholine receptor rich membranes from Torpedo marmorata are shown in Figure B8.3.1. 

Fig. B8.3.1. Fluorescence anisotropy decays at 4 °C of PPL A in phospholipid vesicles (PC PI, 95 5 mol %). B in Torpedo membranes. From the best fit of the /(t) and l (t) components, and by using the wobble-in-cone model, the...

Strambini and Galley have used tryptophan anisotropy to measure the rotation of proteins in glassy solvents as a function of temperature. They found that the anisotropy of tryptophan phosphorescence reflected the size of globular proteins in glycerol buffer in the temperature range -90 to -70°C.(84 85) Tryptophan phosphorescence of erythrocyte ghosts depolarized discontinuously as a function of temperature. These authors interpreted the complex temperature dependence to indicate protein-protein interactions in the membrane. 

The long lifetime of phosphorescence allows it to be used for processes which are slow—on the millisecond to microsecond time scale. Among these processes are the turnover time of enzymes and diffusion of large aggregates or smaller proteins in a restricted environment, such as, for example, proteins in membranes. Phosphorescence anisotropy is one method to study these processes, giving information on rotational diffusion. Quenching by external molecules is another potentially powerful method in this case it can lead to information on tryptophan location and the structural dynamics of the protein. 

K. Kinosita, Jr., S. Kawato, and A. Ikegami, Dynamic structure of biological and model membranes Analysis by optical anisotropy decay measurement, Adv. Biophys. 17, 147-203 (1984). 

The measurement of fluorescence lifetimes is an integral part of the anisotropy, energy transfer, and quenching experiment. Also, the fluorescence lifetime provides potentially useful information on the fluorophore environment and therefore provides useful information on membrane properties. An example is the investigation of lateral phase separations. Recently, interest in the fluorescence lifetime itself has increased due to the introduction of the lifetime distribution model as an alternative to the discrete multiexponential approach which has been prevalent in the past. 

The fluorescence lifetime is sensitive to the environment of the fluorophore, and in membranes this usually means the surrounding fatty acyl chains or the membrane protein interfacial region (see summary in Table 5.3). Generally, the lifetime of membrane-bound fluorophores is rather less sensitive to the types of subtle alterations which are encountered in membranes as compared to the fluorescence anisotropy parameters. The gel-to-liquid crystalline phase transition is a notable exception where most fluorophores show an alteration in lifetime properties. Although, again, the anisotropy (see below) is the most sensitive parameter in this regard, the fluorescence lifetime has been used with considerable success in the study of phase transitions and lateral phase separations. Fluorophores used to yield information on the... 

The first decision to be made in designing an experiment to measure the motional properties of membrane lipids concerns the type of probe molecule. Too often, this choice is made from the point of view of convenience or tradition rather than suitability, although there is now a considerable range of suitable fluorophores from which to choose. The second consideration is the type of measurement to be made. The most detailed and complete motional information is obtained from a time-resolved fluorescence anisotropy measurement which is able to separate the structural or orientational aspects from the dynamic aspects of fluorophore motion. Steady-state anisotropy measurements, which are much easier to perform, provide a more limited physical parameter relating to both of these aspects. 

The rx term is the anisotropy at times long compared to the fluorescence lifetime, whereas in Eq. (5.9) 2 will be long. If there is no rM, then Eq. (5.8) reduces to the familiar Perrin equation for an isotropic rotator. Earlier, some confusion existed in this field since it was not recognized that an rro term was required for the case of membrane lipid bilayers. For the most part, time-resolved anisotropy measurements have a short rotational correlation time and an term. However, it has been recognized that a more adequate description may be to use two rotational correlation times, where the second may be quite long but not infinite as the rm implies/35 36 ... 

At the present time, two methods are in common use for the determination of time-resolved anisotropy parameters—the single-photon counting or pulse method 55-56 and the frequency-domain or phase fluorometric methods. 57 59) These are described elsewhere in this series. Recently, both of these techniques have undergone considerable development, and there are a number of commercially available instruments which include analysis software. The question of which technique would be better for the study of membranes is therefore difficult to answer. Certainly, however, the multifrequency phase instruments are now fully comparable with the time-domain instruments, a situation which was not the case only a few years ago. Time-resolved measurements are generally rather more difficult to perform and may take considerably longer than the steady-state anisotropy measurements, and this should be borne in mind when samples are unstable or if information of kinetics is required. It is therefore important to evaluate the need to take such measurements in studies of membranes. Steady-state instruments are of course much less expensive, and considerable information can be extracted, although polarization optics are not usually supplied as standard. 

There has been considerable interest in using fluorescence anisotropy to detect multiple environments in membranes as with fluorescence lifetimes (see above). For example, if a fluorophore is located in two environments with long and short lifetimes, then the fluorescence anisotropy decay process at longer times after excitation will be dominated by the long-lived fluorescent species. This occurs with parinaric acids, and this situation has been explored for a number of theoretical cases. 60 A similar situation has been found for DPH in two-phase lipid systems by collecting anisotropy decay-associated spectra at early and late times after excitation. 61 Evidence was found for more than one rotational environment in vesicles of a single lipid of it is at the phase transition temperature. It is important to identify systems showing associated anisotropy decays with more than one correlation time, each of... 

If a collisional quencher of the fluorophore is also incorporated into the membrane, the lifetime will be shortened. The time resolution of the fluorescence anisotropy decay is then increased,(63) providing the collisional quenching itself does not alter the anisotropy decay. If the latter condition does not hold, this will be indicated by an inability to simultaneously fit the data measured at several different quencher concentrations to a single anisotropy decay process. This method has so far been applied to the case of tryptophans in proteins(63) but could potentially be extended to lipid-bound fluorophores in membranes. If the quencher distribution in the membrane differed from that of the fluorophore, it would also be possible to extract information on selected populations of fluorophores possibly locating in different membrane environments. 

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