BIOCHEMISTRY - L. Stryer - 1984

VOLUME 1

PART I. CONFORMATION AND DYNAMICS

CHAPTER 10. AN INTRODUCTION TO BIOLOGICAL MEMBRANES

10.13. Lipids and many membrane proteins diffuse rapidly in the plane of the membrane

Introduction/36.html">Biological Membranes are not rigid structures. On the contrary, both Lipids and many Membrane Proteins are in constant lateral motion. The rapid movement of membrane Proteins can be detected by Fluorescence Cell/15.html">Microscopy in the following experimental setup. Cultured human and mouse Cells can be induced to fuse; the resulting hybrid cell is called a heterokaryon. One part of the heterokaryon's Plasma Membrane originates from the mouse cell, and the other from the human cell. Do the mouse and human membrane proteins remain segregated in the heterokaryon, or do they mix? To answer this question, markers—specifically, fluorescently labeled Antibodies—were used and subsequently visualized under a Light Microscope. The antibody against mouse membrane proteins exhibited green fluorescence, while the antibody against human membrane proteins showed red fluorescence (Fig. 10.26). In the newly formed heterokaryon, one half of the surface fluoresced green and the other red. However, in less than an hour (at 37°C), the green and red fluorescent regions became completely intermixed. This experiment demonstrates that a membrane protein can diffuse a distance of several microns in about 1 min.

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Fig. 10.26. Schematic representation of the fusion of a mouse cell and a human cell, followed by the diffusion of membrane components in the plane of the membrane. A few hours after fusion, the green and red fluorescent markers are completely intermixed

A more general and quantitative method for measuring the lateral diffusion of membrane components in intact cells is fluorescence recovery after photobleaching (FRAP) (Fig. 10.27). In this technique, a specific cell-surface component is specifically labeled with a fluorescent chromophore, and a small area of The Cell surface (~ 3 μm²) is focused under a fluorescence microscope. The fluorescent molecules in this area are then destroyed (bleached) by a very intense laser beam. The rate of fluorescence recovery in the bleached area is then monitored using very low-intensity light to prevent further photobleaching. If the labeled component is mobile, the bleached molecules will diffuse out of the observed area and be replaced by fluorescent ones, leading to an increase in fluorescence intensity. The rate of recovery of the initial fluorescence level depends on the lateral mobility of the fluorescently labeled component, which can be expressed in terms of the diffusion coefficient D. The average distance s (cm) traversed in the plane in time t (s) depends on D (cm² • s^-1) According to the equation

_s = (4 Dt)^1/2.

Fig. 10.27. Fluorescence recovery after photobleaching (FRAP) method. A — fluorescence of a labeled cell surface component in a small illuminated area of the cell. B — fluorescent molecules are bleached by an intense flash of light. C — as bleached molecules diffuse out of the illuminated area and unbleached ones enter, fluorescence recovers to its initial level. D — the rate of recovery depends on the diffusion coefficient

The diffusion coefficient of lipids in various membranes is about 10^-8 cm² • s^-1. Thus, a phospholipid molecule diffuses an average distance of 2 • 10^-4 cm, or 2 μm, in 1 s. This means that a lipid molecule can travel from one end of a bacterial cell to the other in 1 s. The experimentally determined value of the diffusion coefficient indicates that the viscosity of membranes is about 100 times greater than that of Water, closely resembling the viscosity of olive oil.

In contrast to lipids, proteins are highly heterogeneous with respect to their lateral mobility. Some proteins are almost as mobile as lipids, whereas others are virtually immobile. For instance, the photoreceptor protein rhodopsin is highly mobile, with a diffusion coefficient of 4 • 10^-9 cm² s^-1. On the other hand, Fibronectin (a peripheral glycoprotein involved in cell-substrate interactions) has a diffusion coefficient D of less than 10^-12 cm² s^-1. The low mobility of some proteins may be due to their anchorage to submembrane cytoskeletal structures.

10.14. Membrane proteins do not undergo transverse movement across bilayers

In contrast to their movement in the plane of the membrane, the spontaneous translocation of lipids from one membrane surface to the other is very slow. The movement of a molecule from one membrane surface to the other is termed transverse diffusion (or "flip-flop"), whereas the diffusion of molecules in the plane of the membrane is called lateral diffusion. Direct measurement of the transverse diffusion of phospholipid molecules in phosphatidylcholine vesicles was carried out using electron paramagnetic Resonance; it was found that a phospholipid molecule flips from one side of the bilayer to the other only once in several hours (the experiment is described at the end of the chapter in the "Problems" section, item 5). Thus, the transverse diffusion of a phospholipid molecule across a distance of 50 Å takes about 10⁹ times longer than lateral diffusion over the same distance.

The free-energy barrier for the transverse diffusion of protein molecules is even larger than that for lipids, because proteins have much more extensive polar regions. No transverse diffusion of proteins has been detected. Consequently, membrane Asymmetry is preserved for long periods.

10.15. The fluid mosaic model of biological membranes

In 1972, S. Jonathan Singer and Garth L. Nicolson proposed the Fluid Mosaic Model, which describes the general Organization of biological membranes. According to this model, membranes are two-dimensional solutions of oriented Globular proteins and lipids (Fig. 10.28). A wealth of experimental evidence Supports this model. The key Features of the fluid mosaic model are as follows.

Fig. 10.28. The fluid mosaic model

1. Most of the membrane Phospholipids and Glycolipids are organized in a bilayer. The Lipid Bilayer plays a dual role, serving both as a solvent for integral membrane proteins and as a permeability barrier.

2. A small fraction of Membrane Lipids is specifically bound to certain membrane proteins and is likely essential for their function.

3. Membrane proteins diffuse freely in the lateral direction within the lipid matrix, but they cannot undergo transverse movement, i.e., from one surface of the membrane to the other.