the control of membrane organization by electrostatic forces

Bioscience Reports 2, 1-13 (1982) 1 Printed in Great Britain

The con t ro l of membrane organizat ion by e l e c t r o s t a t i c fo rces

Review

3. BARBER

Department of Pure and Applied Biology, Imperial College, London SW7 2BB, U.K.

The view that biological membranes are dynamic structures has developed from a range of different types of studies. As a consequence the fluid-mosaic model of Singer and Nicolson (1) is now well established and the task at hand is to fully understand how changes in organization of membrane components control functional activity. In this short review I want to focus at tent ion on non-specific long-range forces which act on protein complexes embedded in the lipid matrix of biological membranes and consider these forces in terms of prote in- protein and membrane-membrane interactions. Discussion will include factors which may control lateral movements of proteins and how such movements could play a role in encourging strong interactions between adjacent membrane surfaces, I will not dwell in depth on the role of lipids in these processes but rather concent ra te on the importance of p r o t e i n d i s t r i bu t ion in d e t e r m i n i n g membrane conformation. To emphasize the concepts put forward I will call heavily on experimental observations resulting from studies using isolated thylakoid membranes of higher plant chloroplasts. I do this, not only because this membrane system is the subject of my own research effor t , but because it has many unique properties which makes it an ideal candidate for elucidating the factors which control conformational changes commonly observed with a wide range of biological membranes.

A br ie f survey of la teral prote in di f fus ion in t e r m s of funct ion and m e m b r a n e - m e m b r a n e in terac t ion

The realization that integral proteins of biological membranes can readily undergo lateral diffusion stems back to about I0 years ago, when it was shown that surface antigens of mouse and human cultured cells were able to intermix when fusion was induced to form hetero- karyons (2). Since then it has been shown by a variety of techniques t h a t the l a t e r a l diffusion of proteins occurs in a wide range of membranes : rhodopsin in pho to r ecep to r membranes (3,4); lectin r ecep to r s ( p a r t i c u l a r l y for Concanaval in A) in myoblas ts (5-7), f ib roblas t s (8-13), myotubes (14), glia (15), and neurons (15,16); surface antigens in mast cells (17) and mouse eggs (18); acetylcholine receptors in myoblasts and myotubes (14,19); hormone receptors in f ib rob las t s (20,21); and integral proteins in erythrocytes (22-25), thylakoid membranes (26-28), mitochondrial membranes (29), and a number of other membranes (30).

�9 1982 The Biochemical Society

2 BARBER

Of the many experimental approaches adopted to detect lateral protein diffusion, the most powerful have been freeze-fracture electron microscopy (31) and laser photobleaching recovery (32). The latter technique involves attaching a fluorescence marker to the membrane protein (particularly convenient for lectin, hormone, and acetylcholine receptors) and subjecting a small area of the membrane (usually a spot of about 3 pro, but also see ref. 33) to an intense flash from a focused laser. The result is to bleach the fluorochrome in the selected area and then monitor the fluorescence rise in this area as f luorescence- label led proteins diffuse in. Thus i t is possible to calculate diffusion coefficients which usually range, for physiological temperatures, from I0-9 to i0 t2 cm 2 sec-t.

The functional significance of lateral protein diffusion is becoming clear for a number of systems, particularly those associated with hormone-receptor triggering and cell-surface immunochemistry (3#-36). Such movements may also control membrane permeability (37), in terac t ions between electron transport components in mitochondria (38), and adenylate cyclase act iv i ty in growth control (39,#0). Often associated wi th these various functional responses is clustering of membrane proteins to form domains (21,35). Also protein aggregation within membranes can be triggered in a number of other ways, such as pH changes (23,37), changing electrolyte levels (# l ) , mild action of pronase and trypsin (#2,#3), treatment with lectins (5,##), and by temperature changes (##-#6). Reorganization of membrane proteins in these various ways will bring about changes in the properties of local regions on the membrane surface. Therefore i t is not surprising that processes involving interactions between adjacent membrane sufaces, such as fusion and formation of appressed lamellae, are usually linked with lateral protein movement and domain formation (#7,#8).

In the case of membrane fusion, Poste and Allison (#9) have proposed that the associated redistribution and aggregation of intramembraneous particles might provide a mechanism whereby areas of membrane become devoid of integral proteins, with fusion taking place between the protein-free areas on adjacent surfaces. This concept has gained support from studies on a wide range of systems including virus-induced and chemically induced fusion (#g,50). This proposal is, however, not applicable to those cases where close interaction occurs between adjacent membranes without fusion. Examples of this type of membrane-membrane interaction are t ight and gap junctions, where the appressed regions usually have characteristic arrays of aggregated membrane particles (51). Presumably the intramembraneous proteins or glycoproteins sequestered in the junctional regions impose surface characteristics which encourage membrane-membrane interaction but prevent fusion.

Forces of in te rac t ion

The brief introduction given above leads to the broad conclusion that biological membranes have heterogeneity in their structure and that they are dynamic systems. Thus their organization will be determined by forces of interaction between adjacent components within the membrane, and also between adjacent membrane surfaces.

ELECTROSTATIC CONTROL OF MEMBRANE ORGANIZATION 3

Such forces will exert their influence on membrane structure especially if the lipid matrix is sufficiently fluid to allow reorganisation of integral proteins to occur under different conditions.

For the i n t e r a c t i o n be tween in tegra l membrane proteins the following should be considered:

(a) entropy of mixing; (b) protein-protein interaction; (c) protein-lipid interaction.

The en t ropy of mixing should favour a random distribution of proteins throughout the available membrane area, but in practice such a randomization will be determined by the other forces of interaction be tween the various components . Interaction between membrane proteins could be both specific (short-range) and non-specific (long- range). Specific interaction may occur between well-defined protein components, while non-specific thermodynamic interactions will a f fec t all the proteins present. Only non-bonding long-range forces will be considered in this article~ since it focuses at tent ion on a more general biophysical property of membrane structure. Also the discussion will be restricted to protein-protein interaction, since the role of lipids in r egu l a t i ng membrane o rgan iza t ion has been the subject of much discussion in recent years (52,53). There are two major types of l ong- range non-spec i f i c forces expected to exist between integral membrane proteins and thus determine their spatial relationships (see Fig. 1 ):

(i) e lectrostat ic forces (coulombic and dipole); (ii) van der Waals forces (non-bonding electrodynamic). The electrostat ic interactions can be both repulsive and at t ract ive,

but at physiological pH the exposed segments of membrane protein o f t e n ca r ry net nega t ive e l ec t r i ca l charge, so that electrostat ic repulsion is more usual except perhaps for some specific cases. The existence of net electrical charge on the surface proteins gives rise to a d i f fuse ionic 'double l ayer ' in the adjacent medium (5#,55). Theoretical t rea tment of the properties of this layer and the associated forces of interaction can, at the f irst approximation, be tackled by using the Poisson-Boltzmann relationship. Addition of electrolytes to the medium reduces electrostatic repulsion, because of the increase in charge screening. Similarly~ lowering the pH of the medium to the isoelectric point of the surface groups giving rise to the net charge wi l l reduce the surface charge density close to zero so that the electrostatic repulsion is minimized. Therefore, either or both addition of salt and lowering of the pH could give rise to a decrease in the distance between integral proteins as depicted in Fig. I . However, in the case of charge screening the distance of approach is restricted due to the increase in coulombic repulsion as the two charged surfaces approach each other~ a fact which is expressed in classical potential energy distance curves (see Fig. 2) similar to that of the Derjaguin- Landau-Vervey-Overbeek (DLVO) theory of charged colloids (56,57). These ideas not only hold for interactions between adjacent integral membrane proteins but may be applied in general terms for the in te rac t ion between extrinsic and intrinsic membrane proteins and between adjacent membrane surfaces (5%55,5g). If, of course, the addition of electrolytes brings about ion binding to the membrane, then this will also modify the surface charge density and affect the balance

4 BARBER

A, Fr .~ ELECTROSTATIC REPULSION

F a ~ VAN DER WAALS ATTRACTION

+ / ~WER PH

B, ~ Fr ,~_ Fa ~v---- . ---

INCREASED ELECTROSTATIC SCREENING OF SURFACE CHARGES

C,

NEUTRALIZATION OF SURFACE

CHARGES BY PROTONATION

Fig, i. A diagrammatic representation of how changes in the balance of long-range non-specific electrostatic (coulombic) and electrodynamic (van der Waals) forces acting between adjacent intrinsic membrane proteins can affect their spatial separa- tions. Reduction of the coulombic repulsion either by salt addition (electrostatic screening) or by adjusting the pH to the isoelectric point of the surface electrical charges (electrostatic neutralization) will) as long as the lipid matrix is sufficiently fluid) give rise to protein aggregation in the lateral plane of the membrane. The packing of the aggregated protein complexes will be greatest in the case of electrostatic neutralization because of the total removal of coulombic repulsion at short distances.

of the opposing forces. It would be dangerous to ignore this possibility because it has been shown that certain inorganic cations can specifically interact with the charged groups of phospholipids (58a). Thus it is very important to distinguish between e lect ros ta t ic screening (surface charge density remains constant) and ion binding (surface charge density modified) when considering changes in coulombic forces due to increasing salt levels (54).


Polential energy l

c 1 ~,Potential energy barrier 0

r ~

,

7 I p! mary minimum

--~ Distance

Secondary minimum

Fig. 2. Schematic potential-energy curve repre- senting the interactions of electrically charged surfaces as a function of distance between them. Lowering the potential-energy barrier by addition of salts (shown as dotted curve) will favour aggregation at distances corresponding to the secondary minimum. This concept is the basis of the Derjaguin-Landau-Vervey-Overbeek (DLVO) theory developed to explain coagulation of charged colloids (56,57). The deeper the secondary minimum and the lower the potential energy barrier~ the more likely that aggregation will occur. Aggregation involving the secondary minimum is usually reversible.

Understanding the nature of the van der Waals forces involved in membrane organizat ion is more diff icult. Clearly, electrodynamic i n te rac t i on wi l l occur between components in the membrane and between adjacent membranes. The theory of van der Waals forces applied to macroscopic systems, including application of the Hamaker method to the L i f s h i t z theory of interaction within and between membranes~ has advanced considerably in recent years. The names Parsegian~ Ninham~ Nir, Richmond, Israelachvili, and Vanderkooi, as well as others, come readily to mind (5g-63). Estimating the relative magnitudes of the van der Waals forces for protein-protein, protein- lipid, and membrane-membrane interaction is a formidable task. In genera% at long distances, these interactions are a t t rac t ive and tend to oppose long-range coulombic repulsion (see Fig. 1).

6 BARBER

Prec i se ly how prote in componen t s i n t e r a c t w i t h i n biological membranes or how two adjacent membranes will respond to each other 's presence will be determined by the special features of the membrane involved. However, my own analyses of phenomena which occur in chloroplast membranes emphasize the above ideas and indicate that general physical concepts can be applied to biological membranes be fo re needing to r e t r e a t to specific 'chemical ' mechanisms, an a r g u m e n t also pioneered by Vanderkoo[ (58) and Gingell (g6). Moreover, studies with chloroplast thylakoid membranes indicate that l a t e r a l p ro te in movemen t s and aggregat ion can occur without a concomitant change in phase properties of the lipid matrix, empha- sizing the importance of protein-protein interaction in regulating the conformational changes.

Ch lo rop la s t thy lako id membranes

General feature

The thylakoids of higher plant chloroplasts are a membrane system in which the ear ly stages of photosynthesis occur (64965). This m e m b r a n e contains chlorophyll-protein complexes and other protein complexes which function to convert light energy into chemical energy. The main processes occurring are (i) photon capture and transfer to a photochemical reaction centre; ( i i) electrical charge separation and stabilization within the reaction centre; and ( i i i ) s econdary electron flow involving redox changes. Both intrinsic and extrinsic proteins are involved. Overall electrons are extracted from water and passed to a low-potential redox species capable of reducing NADP. During this electron transfer process, ATP is synthesized, so that this high-energy compound together with NADPH are the chemical products of the light-driven nrlembrane-mediated processes. The transfer of electrons f rom H20 to NADP requires the cooperation of two photosystems ca l l ed p h o t o s y t e m one (PSI ) and pho tosys t em 2 (PS2). Each photosystem has its own light-harvesting pigment array which serves as an antenna to the reaction centre. There are many hundreds of antenna pigment molecules per reaction centre and these pigments are embedded in proteins which are the major integral complexes of the thylakoid membrane. Chlorophyll a is common to both photosystems as an antenna pigment, but only the chlorophylls of P52 fluoresce significantly at room temperature.

Sensitivity of thylakoid membrane organization to ionic conditions

If the thylakoid membrane is isolated and suspended in a medium of ionic content which has poor electrostat ic screening of surface negative charges, the PS2 and PSI pigment proteins seem to randomize in the plane of the membrane, as indicated diagrammatically in Fig. 3. Under these conditions, energy is transferred from the PS2 chlorophyll antenna to that of PSI (probably by a Forster mechanism) and the overall fluorescence from the membrane is at a low level (because the PSI chlorophyl ls ac t as fluorescence quenchers). Moreover, poor e lectrosta t ic screening inhibits membrane-membrane interactions and


Good energy tronsfer

High fluorescence

ATERAL

salt PIGMENT - PROTEIN

DIFFUSION

time le

Fig. 3. A diagrammatic representation of how altering the electrolyte composition of medium so as to change the screeing of surface electrical charges brings about lateral diffusion of integral pigment- protein complexes in the chloroplast thylakoid membrane. The protein complex of photosystem two (PS2) including the light-harvesting chlorophyll a/chlorophyll b protein (shown as an unshaded particle) is postulated to carry a low net electrical charge on its exposed surface 9 which would explain the aggregation in regions where close membrane interaction can occur (strong van der Waals interactions between adjacent complexes and between adjacent membrane surfaces in the absence of significant short-range coulombic repulsion). The pigment-protein complexes of photosystem one (PSI) are shown as black circles and postulated to carry net negative charge on their exposed surfaces. Because of this they aggregate less readily on addition of salt and are excluded from those membrane regions where the coulombic repulsion is sufficient not to favour membrane appression. Under low-salt conditions 9 coulombic repulsion is large~ so as to prevent aggregation and membrane appression; this condition leads to randomization of the PS2 and PSI proteins in the plane of the membrane. The change from the randomized to the aggregated condition is reversible and can be readily monitored by detecting changes in the yield of chlorophyll fluorescence which reflects alterations in the degree of energy transfer from the PS2 to PSI complexes as shown (see 68).

8 BARBER

appressed regions are not observed. Addition of appropriate ionic species wi l l decrease the coulombic repulsion between the various charged surfaces and i t is visualized that a reorganization occurs as indicated in Fig. 3. The formation of domains as shown in Fig. 3 involves the lateral diffusion of the two types of pigment-protein complexes to different parts of the membrane, so that energy transfer from PS2 to PSI is reduced and the fluorescence increases. Thus the k ine t ics of f luorescence rise is indicative of the diffusion rates involved and can be compared with the fluorescence rise observed in a laser photobleaching experiment mentioned earlier (32). The forma- t ion of two d i f fe ren t protein domains mean that there is now heterogenei ty in the surface propert ies of the membrane, with membrane appression occurring in regions of aggregated PS2 protein complexes. For close membrane interact ion to occur (about 4 nm as viewed in the electron microscope), the net surface charge density must be low (66,67), so that i t is argued that the exposed segments of the PS2 chlorophyll-protein complex is close to electroneutrality while the PSI protein complex has an exposed surface which carries a net negative charge density of sufficient size to prevent membrane appression (27,68).

There is much evidence for this postulated salt-induced reorganization of thylakoid membranes, including:

( i) I t is dependent only on electrostatic parameters, being closely correlated with the space charge density immediately adjacent to the membrane surface. That is, i t is brought about by a wide range of inorganic and organic cations with strong dependency on their valency rather than their chemical nature (27,68-71). ( i i ) The f l u i d i t y of the l ip id matrix is an important factor. Lowering the temperature, introduction of cholesterol into the lipid matrix, or ageing reduces the f luidi ty of the membrane and inhibits the rate of lateral protein diffusion (26,72). ( i i i ) The reorganization can be seen by freeze-fracture electron and fluorescence microscopy (28,41,73,74). ( iv) I t is almost certainly not due to ionic binding but to electrostatic screening. Ionic binding, like protonation, does not give rise to the domain formation of the type indicated in Fig. 3 but allows extensive membrane appression to occur without selective lateral protein movements (54,68). (v) Biochemical and spectroscopic analyses of the appressed and non-appressed lamellae, with reference to complete randomized membranes, indicates loca l izat ion of d i f fe ren t pigments and funct iona l act iv i t ies in the appressed and non-appressed regions (75-78) . Thus the model presented in Fig. 3, supported with the above

evidence, indicates that lateral diffusion and reorganization of integral protein components within the thylakoid membrane, as well as changes in m e m b r a n e - m e m b r a n e interaction, are controlled by non-specific long-range coulombic interactions which counterbalance the a t t rac t ive van der Waal forces. The occurrence of a high protein-to-lipid ratio for the appressed membranes (79) is in accordance with the require- me n t for an i n c r e a s e in the van der Waals interactions between adjacent membranes in these regions (66), while the lower protein- to-lipid ratio of the non-appressed membranes leads to a less rigid


environment having significant fluidity (79). Recently we have been able to use the kinetics of the salt-induced fluorescence rise to crudely est imate lateral diffusion coefficients for the pigment-protein movements: the values obtained ranged from 2 x 10 -12 cm 2 sec -t at lO~ to 2 x 10 - l l cm 2 sec -I at 27~ (80).

C o n c l u s i o n

There seems l i t t l e doubt that in the case of the thylakoid membrane, r e o r g a n i z a t i o n of protein complexes by lateral diffusion is control led by long-range non-specific interactions. This finding has two important implications:

(1) For e luc ida t ing the mechanism of protein aggregation in b iological membranes , as well as for under s t and ing membrane- membrane interaction (appression and fusion), it should be borne in mind that it may not be necessary to invoke specific short-range 'chemical ' or 'physical' processes or place importance on thermally induced lipid-phase transitions.

(2) The concep t tha t spa t ia l relationships between membrane p ro te in complexes are controlled by the electrostat ic and electrodynamic interactions emphasizes the importance of (a) the surface e l e c t r i c a l charge characteristics, (b) the ionic nature of the sur- rounding medium, and (c) changes in van der Waals interactions. Recognition of these factors focuses attention on possible regulatory mechanisms involving changes in membrane organization. For example, reorganization may be under the control of the ionic pumps which a l t e r the electrostat ic screening condition adjacent to the cha rged surfaces involved. Perhaps of more importance is the possibility of regulating the surface charge density. Such a possibility could be achieved by, say, phosphorylating groups on the exposed surface of an i n t eg ra l p ro te in . This type of phosphorylation is well known for membrane systems and is linked closely with their functional activities (136). The likelihood of protein phosphorylation playing a role in bringing about l a t e r a l reorganizat ion of membrane components is reinforced by studies with thylakoid membranes. It is a fact that the PS2 l i g h t - h a r v e s t i n g chlorophyll protein undergoes controlled phosphorylation/dephosphorylation which seems to be related with an in vivo r e g u l a t o r y process of energy transfer from the PS2 antenna chlorophylls to those of PS1 (81-8#). In this way the excitation of the two photosystems is maintained at optimal levels for different lighting conditions (55,83~85). The concept of surface phosphorylation controlling the lateral organization of integral protein complexes within the chloroplast mmembrane is shown in Fig. #. Thus the mechanism suggested indicates that protein phosphorylation brings about a similar, but not so extensive~ change in membrane organization observed, by varying ionic levels (see Fig. 3 for comparison).

Finally~ changes in both electrostat ic and van der Waals interactions will occur if the characterist ics of the integral proteins are altered by~ for example, binding of additional proteins~ formation of d imers , or t r e a t m e n t with p ro tease . Accepting that long-range a t t rac t ive and repulsive forces are important~ then such changes must lead to a reshuffling o f protein components within the membrane and

I0 BARBER

Controlled Intermixing Qf PS1 ond pS2 by Phosp.horylotion

PS2~PSI energy tronsfer poor.

MQximum membrane oppression.

Maximum fluorescence yield.

Dephosphorylotion Phosphorylotion

. . . . . . r - . f ~ ~ ~ PS2~PSI energy tronsfer increosed.

t~O~:~]r'a~l~,~!l~'c~p:' p " Decreose in oreo o| membrone oppression.

O,',~ql[~C~ '~ " . . . . . . " Lowering of fluorescence yield.

Fig. 4. The concept of increasing the net electrical-charge density of some of the photosystem-two (PS2) complexes by surface phosphorylation. As a consequence, the phosphorylated complexes are excluded from the appressed membrane regions and become more intimate with the photosystem-one (PSI) complexes in the non-appressed region. This results in an increase in energy transfer from PS2 to PSI and thus a corresponding lowering of the fluorescence yield. The model also predicts a change in the extent of the appressed area after phosphorylation has occurred. The process can act as a control mechanism to regulate energy transfer from PS2 to PSI (55). The symbols used for the PS2 and PSI protein complexes are the same as those in Fig. 3.

affect membrane-membrane interactions. I t is therefore not surprising that lateral protein movements and aggregation are often observed when antibodies, hormones , and lectins bind to receptor proteins or when membrane junctions form or fusion occurs.

Acknowledgements

I would l ike to thank the Science and Engineering Research Council and the Agr icul tural Research Council for f inancial assistance to allow experiments to be done to support the concepts presented in this art ic le.


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