Lateral diffusion phospholipids

The attachment of pyrene or another fluorescent marker to a phospholipid or its addition to an insoluble monolayer facilitates their study via fluorescence spectroscopy [163]. Pyrene is often chosen due to its high quantum yield and spectroscopic sensitivity to the polarity of the local environment. In addition, one of several amphiphilic quenching molecules allows measurement of the pyrene lateral diffusion in the mono-layer via the change in the fluorescence decay due to the bimolecular quenching reaction [164,165]. 

Two principal routes of passive diffusion are recognized transcellular (la —> lb —> lc in Fig. 2.7) and paracellular (2a > 2b > 2c). Lateral exchange of phospholipid components of the inner leaflet of the epithelial bilayer seems possible, mixing simple lipids between the apical and basolateral side. However, whether the membrane lipids in the outer leaflet can diffuse across the tight junction is a point of controversy, and there may be some evidence in favor of it (for some lipids) [63]. In this book, a third passive mechanism, based on lateral diffusion of drug molecules in the outer leaflet of the bilayer (3a > 3b > 3c), wih be hypothesized as a possible mode of transport for polar or charged amphiphilic molecules. 

Phospholipids, which are one of the main structural components of the membrane, are present primarily as bilayers, as shown by molecular spectroscopy, electron microscopy and membrane transport studies (see Section 6.4.4). Phospholipid mobility in the membrane is limited. Rotational and vibrational motion is very rapid (the amplitude of the vibration of the alkyl chains increases with increasing distance from the polar head). Lateral diffusion is also fast (in the direction parallel to the membrane surface). In contrast, transport of the phospholipid from one side of the membrane to the other (flip-flop) is very slow. These properties are typical for the liquid-crystal type of membranes, characterized chiefly by ordering along a single coordinate. When decreasing the temperature (passing the transition or Kraft point, characteristic for various phospholipids), the liquid-crystalline bilayer is converted into the crystalline (gel) structure, where movement in the plane is impossible. 

To diffuse rapidly in the plane of the membrane (lateral diffusion), a molecule must simply move around in the lipid environment (including the polar head groups). It need not change how it interacts with phospholipids or with water since it is constantly exposed to pretty much the same environment. Lateral diffusion can be slowed (or prevented) by interactions between membrane proteins and the cellular cytoskeleton. This spatially restricts a plasma membrane protein to a localized environment. 

Alkyl chain heterogeneities cause cell membrane bilayers to remain in the fluid state over a broad temperature range. This permits rapid lateral diffusion of membrane lipids and proteins within the plane of the bilayer. The lateral diffusion rate for an unconstrained phospholipid in a bilayer is of the order of 1 mm2 s 1 an integral membrane protein such as rhodopsin would diffuse 40nm2 s 1. 

In the fluid state, the lateral diffusion coefficient of lipids in the bilayer structure is 0( 10 1 ) m2 s-1 (the symbol O is used to indicate order of magnitude). Interestingly, it has been shown that the diffusion coefficients of phospholipids may differ greatly from the inner to the outer leaflet of the biomembrane layer [4,5]. Again, this is related to the differences in chemical... 

Lateral diffusion of phospholipids in model membranes at ambient pressure has been studied over the years by a variety of techniques including fluorescence recovery after photobleaching (FRAP), spin-label ESR, pulse field gradient NMR (PFG-NMR), quasielastic neutron scattering (QENS), excimer fluorescence and others.In general, the values reported for the lateral diffusion coefficient (D) range from 10 to 10 cm /s in the... 

There is an abrupt decrease in the lateral diffusion coefficient of DPPC upon the phase transition from the GI phase to the Gi phase. This is because the acyl-chain region is being packed even more efficiently in the Gi phase than in the GI phase, and the hydrocarbon volume in the Gi phase is smaller than in the GI phase. Also, in the Gi phase, the lipid acyl-chains from the opposing bilayer leaflets interdigitate. In order for a phospholipid molecule to diffuse it has to circumvent the nearby interdigitated molecules which hinder diffusion. 

Falck, E., Patra, M., Karttunen, M., Hyvonen, M.T., Vattulainen, I. Lessons of slicing membranes interplay of packing, free area, and lateral diffusion in phospholipid/cholesterol bilayers. Biophys. J. 2004, 87, 1076-91. 

A very brief description of biological membrane models, and model membranes, is given. Studies of lateral diffusion in model membranes (phospholipid bilayers) and biological membranes are described, emphasizing magnetic resonance methods. The relationship of the rates of lateral diffusion to lipid phase equilibria is discussed. Experiments are reported in which a membrane-dependent immunochemical reaction, complement fixation, is shown to depend on the rates of diffusion of membrane-bound molecules. It is pointed out that the lateral mobilities and distributions of membrane-bound molecules may be important for cell surface recognition. 

The rates of lateral diffusion of phospholipids in lipid bilayer membranes, and in biological membranes, were first measured using spin-labeled lipids.26 50 10 11 9 In general, these rates have been determined by incorporating spin-labeled lipids such as (V) and (VI) in phospholipid bilayers, or multilayers. The paramagnetic resonance spectra of labels such as (V), as well as the nuclear resonance spectra of other lipids in membranes containing (V), depend on the concentration c of the label in the membrane and the rate of lateral motion of the lipids. Two methods... 

Fig. 4. Schematic representation of transient method employed by Devaux and McConnell9 to measure the rates of lateral diffusion of phospholipids in model membranes. The upper diagram represents a concentrated patch of labels at the beginning of the experiment, time f = 0. At later times f>0, the molecules diffuse laterally, as shown in the lower two drawings. The paramagnetic resonance spectra depend on the spin-label concentration in the plane of the membrane, and an analysis of the time dependence of these spectra yielded the diffusion constant. [Reprinted with permission from P. Devaux and H. M. McConnell, J. Am. Chem. Soc., 94, 4475 (1972). Copyright by American Chemical Society.]...

A second steady-state method involves the analysis of the broadening of the nuclear magnetic resonance spectra of phospholipids in bilayers containing low concentrations of spin-labeled phospholipids. A theoretical analysis of the relation between this line broadening and diffusion rates has been given by Brulet and McConnell.3 [In this paper (6) is not correct the subsequent equations are nonetheless correct. For an alternative derivation, see Brulet.2] In this paper it is shown that a number of measurements of nuclear relaxation rates T71 of nuclei in phospholipids are consistent with lateral diffusion constants in the range 10 7 to 10 R cm2/s. 

At the present time, the rates of lateral diffusion of phospholipids and membrane proteins in the solid phase of pure phospholipids is not known. It is hoped that such diffusion constants can be obtained by one of the transient methods mentioned earlier. It is likely that these diffusion rates will be found to be quite low. 

A more interesting problem from both the experimental and theoretical point of view is the lateral diffusion of phospholipids in mixtures of lipids, when both solid and fluid phases coexist. At least three questions arise in connection with this problem. (1) What is the rate of lateral diffusion of phospholipids in solid solution domains (2) To what extent do solid solution domains act as obstacles to the lateral diffusion of lipid molecules in fluid domains (3) To what extent are there composition and density fluctuations present in fluid lipid bilayers, and to what extent do these fluctuations affect lateral diffusion Let us consider these questions one at a time, bearing in mind that these questions may to some extent be interrelated. 

Obviously, long-lived solid phase domains may provide severe obstacles to the lateral diffusion of fluid phase phospholipids, if an appreciable fraction of the lipids are in the solid solution phase. The magnitude of this effect may be affected by the lifetime of the solid phase domains, mentioned earlier. 

As indicated in my report, we now know the rates of lateral diffusion of phospholipids in lipid bilayers in the fluid state, and in a few cases the rates of lateral diffusion of proteins in fluid lipids are also known. At the present time nothing is known about the rates of lateral diffusion of phospholipids in the crystalline, solid phases of the substances. As mentioned in my report, there are reasons to suspect that the rates of lateral diffusion of phospholipids in the solid solution crystalline phases of binary mixtures of phospholipids may be appreciable on the experimental time scale. Professor Ubbelohde may well be correct in pointing out the possibility of diffusion caused by defects. However, such defects, if present, apparently do not lead to significant loss of the membrane permeability barrier, except at domain boundaries. 

FIGURE 11-16 Motion of single phospholipids in a bilayer, (a) Movement from one leaflet to the other is very slow, unless (b) catalyzed by a flippase in contrast, lateral diffusion within the leaflet (c) is very rapid and requires no protein catalysis. 

The authors reported that the hydrophobic peptide had little influence on the lipid structure, as lipid lateral diffusion rates, lipid conformations, and head group orientations were identical to a neat bilayer. The distance distribution of the phospholipid atoms surrounding the peptide was rather broad, pointing to the absence of special interactions between the peptide and the surrounding phospholipids. 

Ellena, J.F., L.S. Lepore, and D.S. Cafiso (1993) Estimating lipid lateral diffusion in phospholipid vesicles from 13C spin-spin relaxation. J. Rhys. Chem. 97, 2952-2957. 

The FRAP method has been applied to the measurements of molecular lateral diffusion of molecules adsorbed at the interface of equilibrium common thin foam films and of black foam films [39-43], Initially Clark et al. reported FRAP measurement of surface diffusion of the fluorescence probe 5-N(octadecanoyl)aminofluorescein incorporated into foam films stabilised with NaDoS [39]. Then followed the measurements of protein-stabilised foam films where the protein was covalently labelled with fluorescein [40,41], Studies of FRAP measurements of surface lateral diffusion in equilibrium phospholipid common thin foam films and black foam films were also reported [42,43]. 

Lateral diffusion in phospholipid black foam films... 

A very suitable method for measurement of the lateral diffusion of molecules adsorbed at the foam film surfaces is Fluorescence Recovery after Photobleaching (FRAP) ([491-496], see also Chapter 2). Measurements of the lateral diffusion in phospholipid microscopic foam films, including black foam films, are of particular interest as they provide an alternative model system for the study of molecular mobility in biological membranes in addition to phospholipid monolayers at the air/water interface, BLMs, single unilamellar vesicles, and multilamellar vesicles. 

Several studies show that the values of the lateral diffusion coefficient (D, cm2 s 1) in foam films stabilised by phospholipid(s) depend on two main groups of factors. The first is related to the type of the film, its thickness and radius, and the lipid composition of film monolayers. The second is related to the dependence of the surface diffusion within the limits of given film type and composition on the molecular characteristics of the lipid(s) building the film (molecular charge, length, lipid phase, etc.). 

Fig. 3.111. Dependence of lateral diffusion coefficient (D, cm2 s 1) of surface adsorbed fluorophore molecules on phospholipid foam film thickness (h, nm) r = 100 - 500 pm t = 24°C [493].

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