Anisotropy decays membranes

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). 

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...

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). 

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. 

W. van der Meer, H. Pottel, W. Herreman, M. Amclott, H. Hendrickx, and H. Schroder, Effect of orientational order on the decay of the fluorescence anisotropy in membrane suspensions, Biophys. J. 46, 515-523 (1984). 

Order parameters are used to interpret data on order and fluidity of a number of probes in lipid membranes obtained by measurements of fluorescence anisotropy decay 32 Ambiguities in the interpretation of time resolved fluorescence anisotropy measurements in lipid vesicle systems with DPH or TMA-DPH probes are attributed to the unsatisfactory models being used to interpret the data . The solubilisation of diphenylpolyenes in lipid bilayers has been critically examined33. It is concluded that such probes are satisfactory if used at low concentrations. 

Straume M, Litman BJ. Equilibrium and dynamic bilayer structural properties of unsaturated acyl chain phosphatidylcholine-cholesterol-rhodopsin recombinant vesicles and rod outer segment disk membranes as determined from higher order analysis of fluorescence anisotropy decay. Biochemistry 1988 27 7723-7733. 

Quenching studies of protein fluorescence provide answers regarding the accessibility of certain internal or external groups to quencher molecules. Another application concerns the study of associative behavior and properties of proteins and membranes. The rationale is that the fluorescence transition is polarized and this polarization can be exploited in time-resolved analysis and interpreted in terms of the rotation or tumbling motion which in turn is determined by the viscosity and structure of the environment of the fluorescing group. In particular, anisotropy decay studies have yielded a great deal of information on the mobility of natural and artificial membranes and/or the dynamics of proteins as well as small molecules in membranes. For such studies fluorescence lifetime labels that can be attached to proteins or that dissolve in membranes have... 

Fig. 3. Effect of melittin binding to Ca -ATPase in sarcoplasmatic reticulum on the time-resolved anisotropy decay of the membrane probe diphenylhexatriene. (From Voss et al. )...

A typical reailt from these sUidies is that the presence of cholesterol in the membranes results in more hindoed rotational diffusion than in the absence of cholesterol. This can be seen in the experimental anisotropy decays of DPH in DPPC vesicles, as the mole ffaction of cholesterol is increased (Figure 11.10). Similar behavior has been ob-SCTved for DPH in a wide range of phospholipids " and for other meinbrane>bound probes. Such behavior is a genoal feaUire of the anisotropy decays of labeled membranes. 

Hgure 11.10. Anisotropy decays of DPH in DPPC ve ks at 49.5 C, coniaming 0, and 50 mol % cholesteroL At 49.5 "C the MW membranes are above their phase-transition temperature. M iich is near 37 Revised from Ref. 29. 

Figure 11.16. Anisotropy decay ofdansyl-lysine bound to the antigen binding sites of IgE in the absence and in the presence of the membrane receptor. Revised and reprinted, with permission, firom Ref, 74, Copyright 1990, American Chemical Sodeiy.

StudiN of DNA by fluorescence can be traced to the use of dyes lo stain chromatin for fluorescence microscopy. The use of time-resolved fluorescence for DNA dynamics originated with the measurement of anisotropy decays of EB bound to DNA. " These early studies showed an unusual anisotropy decay, similar to that found for DPH in membranes, in which the anisotropy at long times did not decay to zero (Figure 11,24). At that time, the results were interpreted in terms of the angle through which the EB could rotate within the DNA helix. However, more recent... 

At 800 nm we can resolve a 7.6 1.7 ps anisotropy decay (Table 2) that does not correspond to any component in the isotropic signal. Therefore this signal probably results from the migration of the excitation energy in the baseplate. The value for roo of 0.13 0.02 is close to the value of 0.10 expected if the main BChl a transition is circularly distributed in the membrane plane (0 = 90° in the equation above). 

Because the molecule s motion is restricted, the anisotropy does not decay to zero but reaches some final constant value, r . Equation 19 is typically used to model the anisotropy decay of a rod-shaped fluorophore such as l- 4-trimethylammomumphenyl)-6-phenyl-l,3,5-hexatriene (TMA-DPH) embedded in a phospholipid membrane, where it reports on the order and dynamics of the latter. The so-called order parameter S of the membrane is defined as... 

The above properties and phenomena can be assessed with great sensitivity and precision by the measurement of rotational diffusion usually based upon the combined use of polarized excitation and deactivation processes. The faster motions alluded to above are particularly well adapted to the techniques of nuclear magnetic relaxation and fluorescence depolarization, the formalisms for which are extensively documented (references 2 and 3, respectively, and citations therein other chapters in this volume). Optical anisotropy decay measurements with longer time resolution have been very effective in studies of biological and model membrane systems (reviewed in 4-6). 

Figure 5. Effect of anisotropy on flow decay of 0.45pM Tyrann-M/E and conventional membranes with 0.01% Triton X-400 solutions. Relative pore size adjacent to feed 1 = Tyrann-M/E, large 2 — Tyrann-M/E, small 3 = conventional, large 4 = conventional, small.

The decay of the anisotropy for a chrmnophore rotatit between two barriers has been derived by Wahl . The proMem posed by a fluorophore with restricted rotation in an ordered lipid bilayer has been discusred by Kinosita et al. If ro is the initial anisotropy created in tire stem at tinre 0 and r is the final limiting value after long times, then, unlike an isotropic, low viscosity solvent, r /ro will not be zero, but will be a measure of the degrte of confinement of the orientatfon of the label imposed by the architecture of the membrane. The dec in r(t) between th two limits should reflect the degree of wobbling mot ns possible for the fluorophore. 

---