Co-existing lyotropic liquid crystals : commensurate, faceted and co-planar single hexagonal (HII) domains in lamellar photoreceptor membranes

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Co-existing lyotropic liquid crystals : commensurate, faceted and co-planar single hexagonal (HII) domains in

lamellar photoreceptor membranes

S.M. Gruner, K.J. Rothschild, W.J. Degrip, N.A. Clark

To cite this version:

S.M. Gruner, K.J. Rothschild, W.J. Degrip, N.A. Clark. Co-existing lyotropic liquid crystals : com- mensurate, faceted and co-planar single hexagonal (HII) domains in lamellar photoreceptor mem- branes. Journal de Physique, 1985, 46 (2), pp.193-201. �10.1051/jphys:01985004602019300�. �jpa- 00209958�

Co-existing lyotropic liquid crystals : commensurate, ^faceted and co-planar single hexagonal (HII) ^{domains in lamellar}

photoreceptor ^membranes

S. M.

Gruner (1*),

^{K. J.} Rothschild

(2),

^{W. J.}

DeGrip (3)

^{and N. A. Clark}

(4)

(1) Dept. ^of Physics, ^Princeton University, Princeton, ^NJ 08544, ^U.S.A.

(2) Depts. ^of Physics ^and Physiology, ^Boston University, ^{Boston, MA} 02115, ^U.S.A.

(3) Dept. ^of Biochemistry, University of Nijmegen, ^{P.O. Box} 91101, 6500 ^HB Nijmegen, The Netherlands

(4) Dept. ^of Physics, ^{Condensed Matter} Laboratory, University ^of Colorado, Boulder, ^CO 80309, ^U.S.A.

(Reçu ^le 22 juin 1984, accepté ^{le 9 octobre} 1984 )

Résumé. ²⁰¹⁴ Les interactions de domaines hexagonaux ^inverses (HII) ^{avec une} matrice lamellaire (L03B1) ^ont été étudiées dans deux cristaux liquides lyotropes par diffraction X et microscopie électronique. ^{Les domaines} présentent ^une

structure interne qui ^est géométriquement couplée à l’hôte lamellaire : les périodes cristallographiques ^{sont souvent}

commensurables et une couche hexagonale ^est coplanaire ^{avec les} lamelles. L’examen de couches minces au micro- scope électronique ^{révèle une section} de domaines HII ^{limités par} des facettes provenant ^{de la structure} hexagonale.

Les domaines sont en équilibre ^{avec leur hôte et} peuvent ^être élargis ^ou comprimés ^en faisant varier la température

ou la quantité ^d’eau. L’empaquetage hexagonal ^{local est} bidimensionnel; la troisième dimension présente ^souvent

des textures spirales ^et toroïdales accompagnées de défauts et bifurcations. En prenant ^en compte les contraintes

hydrophobes ^et hydrophiles ^{on obtient un modèle} pour la paroi limitant les domaines. L’hypothèse supplémentaire

d’une courbure induite _par la surface, suggère ^{un modèle} pour la croissance épitaxiale des domaines.

Abstract. ²⁰¹⁴ The interactions of inverse hexagonal (HII) domains with a host lamellar (L03B1) matrix have been inves-

tigated ^{in two} lyotropic liquid crystals ^via X-ray diffraction and electron microscopy. The domains exhibit internal structure which is geometrically coupled ^to the lamellar host : the crystallographic repeats ^{are often} commensurate and one hexagonal layer ^is co-planar with the host lamellae. Electron microscope thin-sections reveal the cross-

section of single HII ^{domains to} be limited by facets which arise from the hexagonal structure. The domains are in

equilibrium with the host and can be made to grow ^or shrink by variations in temperature ^or water content. The local hexagonal packing ^is two-dimensional; ^the third dimension often exhibits spiral ^{and toroidal textures accom-} panied by branching ^defects. Consideration of the hydrophobic-hydrophilic constraints leads to a model for the domain wall. Coupled ^{with a} hypothesized surface-induced curvature, this suggests ^a model for the epitaxial growth of the domains.

Classification Physics ^Abstracts 64. 70 M

1. Introduction.

Lyotropic

liquid

crystal phase transitions between lamellar and

hexagonally packed

^{tubular structures}

(La-HII) (5)

^are commonly ^{observed in} lipid-water

mixtures (Fig. 1). Although ^the phase diagrams ^{of a}

number of such

lipid

systems ^{have been}

explored

[4],

very little is known about the interaction between

co-existing La

^and

Hn

domains. The

La-HII

^transition

is of interest to both

physicists

^and

biologists

^because

it lends

insight

^into

cooperative phenomena

^and

interfacial structure in

ampiphilic

systems. ^{In a} system of

co-existing La

^and

Hn

^{domains two} interfaces are

involved : the

lipid-water

interface and the domain wall

separating La

^and

Hn

^volumes.

Normally lipids,

which consist of a

hydrophilic headgroup

^connected

to two

hydrophobic hydrocarbon

polymer ^chains [5], organize ^{in the} presence of water so that the head- groups ^are

hydrated

^{while the}

hydrocarbon

^{chains are}

shielded from _exposure to water. In the

La

phase, ^two planar monolayers of molecules align, tail-to-tail, ^to

form

bilayers

^{which are}

separated by

^{sheets of water}

(Fig.

la). ^{As the water content is} lowered, ^{or the tem-}

Article published online by EDP Sciences and available at http://dx.doi.org/10.1051/jphys:01985004602019300

194

Fig. ^{1. -} a) ^{A schematic} representation ^{of a} cross-section of the La phase. Lipid ^{molecules are shown as a} polar ^head-

group (dark dot) ^{in contact with water} (cross-hatched). ^Two

melted hydrocarbon ^{chains are} covalently ^{linked to each} headgroup. ^The bilayer repeat ^is dL. Proteins, ^which may also be imbedded in the bilayer, ^{are not shown.}

b) ^{In the} HII phase ^the lipid molecules form tubes which surround water cores. The tubes, which extend out of the plane of the paper, pack hexagonally. ^{The tube} layer repeat distance is dH.

perature ^is raised, ^the

lipid monolayers

^{curl into}

long

concentric

cylinders

^{of water and}

lipid

^which

stack hexagonally [1]

(Fig.

lb). ^Note that there is no

continuous transformation of the lipid ^{water interface}

which will _carry

La

^into

Hjp

^We have termed this a

topologically

discontinuous transition to

emphasize

that the

restructuring

^{of the water} interface, ^{and the}

concomitant _exposure of

hydrocarbon

^to water, ^is

likely

^to present

significant

barriers toward the trans-

formation.

The model of the HII phase ^{shown in}

figure

^{1 b is}

derived from X-ray diffraction studies of

randomly

oriented

Hn

^domains [4]. Although ^{this model is}

microscopically

^{accurate it does not address}

questions

about the structure of the domains on a larger ^scale.

Consider, ^for

example,

^the

L(X-Hn phase

co-existence

regions

^{seen in the}

lipid phase diagrams

[4, 5]. ^The

Hit

tubes are not

infinitely

long. ^How do the tube ends terminate against ^a

La

domain ? Since the tubes

comprising

^an Hu ^{domain are}

hydrophobic

^{on their} outside, it follows that such a domain cannot be imbedded in a lamellar

region

without either the _pre-

sence of a

specialized

domain wall or the _exposure of

hydrocarbon

^{to water.} Consequently, it is of interest to ask how

adjacent

^{domains are} delimited and interact. In this article we report ^on the co-existence of

La

^and

Hn

^{domains in}

biologically

^derived

liquid

crystals.

^It is demonstrated that the internal structures of the two domains are

coupled

^{across the domain}

wall _{in ways} which suggest ^{that the}

HII

^is

epitaxially

derived from the

Lcx.

^{It will be seen} that the domain wall is of sufficient _energy ^to allow the

growth

^{of a} single

liquid

crystal ^{domain of}

HII,

with well-defined facets, ^{in a host} lamellar matrix.

2.

ExperimentaL

Two closely ^related

liquid

crystals ^were used in this

study.

The first consists of multilayers ^of

purified

native rod outer segment (ROS) disk membranes extracted from bovine retinas. This

membrane,

^which

has been the

subject

of intensive

biophysical

^{and bio-}

chemical research [6, 7] ^{is a}

primary

^{site of} photon

absorption leading

^to vertebrate vision. The membrane is isolated in the form of closed _sacs, or vesicles, ^whose

composition

is dominated

by

^several

species

^of phos-

pholipid

^{and the}

integral

^membrane

protein, rhodop-

sin. The membrane is washed in distilled water and

ultracentrifugally

sedimented onto a glass ^substrate

via the

isopotential spin dry

technique [8, 9] ^to pro- duce an oriented,

multilayer

^{about 1 cm across}

by

10-20 _ym thick. The second

liquid

crystal ^is

synthe-

sized

by making

reconstituted vesicles of

purified rhodopsin

ⁱⁿ

phosphatidylethanolamine

(PE) ^extract-

ed from hen _eggs (1 ^mole

rhodopsin/80

^moles PE).

(See

[26] ^{for more details on the} rhodopsin

purification

and a reconstitution similar to that described

below.) Rhodopsin

is reconstituted into PE vesicles out of

nonylglucoside

^solution

by rapid

dilution of the

rhodopsin-lipid-nonylglucoside

^{solution to a final}

nonylglucoside

concentration of 3 mM. The dilution

was

performed

^{on ice to} suppress HII formation. The vesicles which spontaneously ^{formed were sedimented}

by centrifugation

(2 h; 100,000 ^x g; 4 °C). These vesi- cles are formed into a

multilayer

^{in the same} way ^as the native membrane

specimens.

^As will be noted below, various data suggest ^{that the}

phase separation phe-

nomenae to be described are not peculiarities ^{of the}

rhodopsin

^{or the}

photoreceptor

^membrane.

Thin sections of reconstituted photoreceptor ^mem-

brane were made

using

^the method of Schindler

et al.

[10].

Samples ^were

prepared

^on glass

microscope

slide

coverslips

^and covered with filter _paper soaked in a solution of 2

% glutaraldehyde

^{and 0.1 M sodium}

cacodylate.

Additional steps ^are described in refe-

rence [10].

In an earlier

study,

^we examined the effect

of hydra-

tion on the phase behaviour and lattice structure of native photoreceptor ^membrane

specimens by

X-ray

diffraction and electron

microscopy

[11].

Hydration

of the

multilayers

^was

isothermally

^varied

by equili-

bration

against

helium of various relative humidities.

Simultaneously,

X-ray diffraction was used to

probe changes occurring

in the lattice structure. The use of a

fast electro-optical ^area X-ray ^detector [12] reduced the time required ^{for an} X-ray exposure ^{to a} few minutes,

thereby facilitating

^the

exploration

^{of the}

phase

behaviour over a wide

humidity

range. At 4 OC, ^and from vacuum

dryness

^{to near} saturation, ^the speci-

mens were found to be

predominantly

^{in a}

La

phase

with a small

co-existing

fraction of

Hn

^{domains. Two}

peculiarities

^{of the}

phase separation

^are

particularly

relevant :

(1) ^The hexagonal ^{lattice of tubes was oriented so}

that the layers ^{of tubes were} always

exactly

parallel

with the

La

planes. ^{In the context of}

figure

1, ^this

means that throughout the bulk of an

Hn

^domain, planes

representing

layers ^{of tubes} (e.g. ^line BB) ^would

be

parallel

^to line AA of the

surrounding La

^matrix.

Moreover, the line width of the

Hn

diffraction was

limited by ^{the narrow} width of the incident beam, ^indi-

cating

^{that the}

H,I

domain contained many, ^{unit cells} and did not consist of only ^{a few} layers ^{of tubes.} Thus,

the

geometrical

organization ^of

lipid

tubes in the centre of the

Hn

^{domains was} influenced

by

^the

La organization

^even though ^{these were at} least several unit cells removed from it.

(2)

^The

La

^and

HII

layer

spacings (distances dL

^and

dH

ⁱⁿ

Fig. 1)

^were often observed to be commensurate with a ratio

of dL : :,dH

^{= 2 : 3} (the

La

^{unit cell, as}

explained

^{in the} Discussion, ^{has 2} bilayers). ^{This is}

dramatically

^{shown in} figure ² which shows the

posi-

tion of 16 orders of Bragg diffraction

spaced

^{on the}

meridian. The

positions

^were measured from the densitometer tracing ^{of an} X-ray ^film exposure of the

multilayer.

Each order on the meridian was

sharp

and of equal ^mosaic

spread.

However, each third order was shown to

actually

^{be a}

superposition

^{of the}

3 K lamellar and (K, 0) hexagonal ^orders (K ⁼ 0, + 1, ...). ^{This was} proven

by

^the presence of off- meridional orders associated with the

hexagonal

Fig. ^{2. - The} positions of 20 consecutive diffracted orders from a native membrane specimen ^{are shown vs. an} integer

order assignment. ^{This data was} taken from an X-ray ^film

exposure [11 ]. The relative error in each datum is approxi- mately equal ^{to the width} of the dots shown. Each 3rd order is actually ^the superposition ^of two commensurate lattices.

The repeat period ^{is 117.3 A} (2 opposed, asymmetric bilayers/

unit cell).

lattice. The lattices were completely commensurate at 50 % ^relative

humidity.

By

changing

^the

humidity

of the

surrounding

gas, the two lattices could be made to diverge, ^since

they

^{swell at different rates}

(Fig.

3).

As the lattices

diverged,

the third order meridional diffraction

split

^{into two} orders; the off-meridional diffraction did not

split

but swelled with one of the

split

meridional components.

The reason

why

the lattices must become incommen- surate at

high

^{or low}

hydrations

^is

readily

understood.

Whereas the lamellar lattice can accommodate an arbi- trary ^water

layer

thickness, ^the

HII

^{lattice cannot}

[13].

At low water fractions, ^the

lipid-water

^{interface must}

bend with a small radius of curvature;

repulsion

between the polar

headgroups

limits this curvature.

At the other extreme, ^{as the water} fraction increases the

lipid

^{tube diameter} grows. The finite length ^{of the}

hydrocarbon

tails limits the diameter of

lipid

^{tubes if}

the

hydrocarbon

^{is to fill the space} between the water tubes. What is not understood is why ^{the rate of}

change

of

spacing

^with respect ^to

hydration

^{for the two}

lattices should

approach

^{one another as the}

spacing

becomes commensurate.

We have now

applied

^electron

microscopy

^{and X-}

ray diffraction to

synthetic PE-rhodopsin liquid

crys- tals and observed similar

phase separation

phenome-

nae. The advantage ^{of electron} microscopy ^{is that}

it allows visualization of individual domains and

yields

information about the domain wall.

Figure

^4a

shows a thin section of the

liquid crystal

^{with the cut}

taken

perpendicular

^{to the} substrate. The fine,

parallel

lines around the

periphery

^{of the}

figure

^{are the stained}

membrane layers viewed in cross-section. The majo-

rity

(> ⁹⁰

%)

^{of the}

micrograph

field contains such

Fig. ^{3. -} Repeat ^{distance vs.} equilibrated ^relative humidity

for a native membrane specimen ^{at 3} temperatures. ^{Each of}

seven X-ray film exposures ^were analysed for the lamellar repeat distance, ² dL ~

120 A,

^{and the} HII ^tube layer repeat (see Discussion for why the lamellar repeat ^{is 2} dL ^{and not} dL). ^The symbols ^show (2/3) dL ^and dH. ^The curves, showing

the variation in spacing ^vs. humidity, ^{were drawn} by eye.

The inset lists the temp. (OC) associated ^{with each} data point

196

Fig. ^{4. -} a) ^Electron micrograph ^{of a} stained thin section of a PE-rhodopsin specimen. ^The parallel lines around the

periphery ^are the membranes in an La ^{phase. The centre}

is an Hn ^domain (see text).

b) ^An optical diffraction pattern ^{of the centre of the} crystal

shown in (a) (left), ^{and of the} L,, ^surround (right).

c) The lattices reconstructed from the optical diffraction of the crystal (left) and of the La phase (right). ^{Distances are}

in A and angles ^{are in} degrees.

lines,

demonstrating

^{that the}

specimen

^is

mostly

lamellar. A small fraction of the field, however, ^stains

darkly;

^{these areas are} often limited

by

straight

edges.

A

particularly

^clear example ^{is shown in the centre}

of

figure

^4a. By ^visual

inspection

^{of the}

original

^micro- graph

negative,

^{one can resolve rows} of low-contrast dots

parallel

^{to each of the} straight

edges

of the dark

region.

^When optical diffraction is

applied

^{to the}

centre of this

region,

^one obtains the diffraction shown in figure ^{4b. From} this, ^{one can} reconstruct the lattice structure shown in

figure

^{4c. If the} dark stained

region

was a «

crystal »

^of

lipid

^tubes

packed

^{on centres as}

in

figure

^4c

(with

^{tubes axes}

approximately perpendicu-

lar to the

plane

^{of the}

paper),

then the included angles

where the

crystal

^{faces meet} would also match those of figure 4a. Measurement of the 3

right-most

^included

angles

^of

figure

^4a

yield

111, 136 ^{and 115°} ± 4°. Given the

uncertainty

^{in the} optical diffraction determina- tion of the lattice repeat, ^the agreement is excellent.

The lattice of

figure

^{4c is not}

perfectly

hexagonal

whereas X-ray diffraction of similar

specimens

always

yields

purely hexagonal ^{lattices. A} hexagonal ^lattice

could

yield

^{that of} figure ^{4c if the} thin section slice

were not

perfectly orthogonal

^{to the}

lipid

^{tube axis}

or if the thin section was

plastically

deformed while

being

^cut.

Optical

diffraction of the lamellar

region

^{in the}

periphery

^of figure ^4a

yields

^a repeat ^{of 81}

A,

^{as shown}

to the

right

ⁱⁿ

figure

^{4c. As with the} native membrane

specimens, the lamellar and

hexagonal

^{domains are}

commensurate (see Fig. 4c) ^{and one}

hexagonal

layer ^is co-planar with the host lamellar lattice. The reconsti- tuted

specimens

^differ from the native

specimens

ⁱⁿ

that the former have one layer ^{of tubes} per

bilayer (dL : dH

^{= 1} 1) whereas the latter

have dL : dH

^{= 2 : 3.}

We know of no other example ^of phase

separation

where the internal

degrees

of freedom of adjacent

domains are so

coupled.

The presence of faceted domain faces

implies

^that

it is

thermodynamically

unfavourable to have a

highly

contorted domain wall. Moreover, ^because the pre- dominant

Hu

^{matrix is}

composed

^{of the} highly hydro-

phobic lipid

tails, ^{it seems}

likely

^{that the domain wall}

is itself a

highly

structured

assembly

of molecules which separates

hydrophobic

^and

hydrophilic

regions.

If this were the _case, then domain walls parallel ^to

adjacent

^lamellae (i.e., ^the

boundary

^{on the} top ^and bottom of the

Hu

crystal ⁱⁿ

Fig.

4a) ^are

likely

^{to be}

different in structure than walls which are non-parallel

to lamellae (i.e., ^the

right-most

crystal ^{faces in}

Fig.

4a).

To illustrate, ⁱⁿ

figure

^{5a and b we} suggest ^how

parallel

and

non-parallel

domain walls

might

be formed. It is

emphasized

that little concrete data

currently

^exists

as to the structure of the domain wall. This distance scale involved does, however, suggest that direct visualization of the domain wall _may be possible ^via

freeze-fracture electron

microscopy

(see, ^for instance,

Fig.

7,

below).

The

La

^and

Hu

^{domains are in}

thermodynamic equilibrium

^{with one} another. As environmental conditions change, ^{one domain} type ^can grow ^at the expense of the other. It is known that the

La-HII equili-

brium can be shifted toward

growth

^{of the}

La

phase by

either

lowering

^the temperature ^or

raising

^the hydra-

tion

(see,

^{for example,}

^Fig.

^{19 of Ref.}

[4]).

^{This can}

readily

be verified for the photoreceptor ^membrane

specimens by observing

^the X-ray diffraction as the temperature ^{of the}

specimen

is varied. It is seen that

(Fig.

6), ^{as the} temperature ^is lowered, ^the

Hu

^diffrac-

tion

disappears

(the temperature ^at which this

happens depends

^{on the} water content of the

specimen).

^Exami-

nation of the

high

angle (4 ^{to 5}

A)

diffraction indicates the

lipid

^to still be in a melted chain (i.e.,

La,

^not

L.)

state. Upon

raising

^the temperature ^the HII ^diffrac-

tion reappears. Note that _upon

raising

^the temperature

the

hexagonal

^{lattice returns} with the former 6-fold

positional symmetry,

indicating

^{that the} layers ^of

Hn

tubes are

again

co-planar ^{with the} La planes. ^The

Fig. ^{5. - Possible} configurations ^of HII-La domain walls

are shown (a) parallel ^{to the} La bilayers ^and (b) ^not parallel

to the bilayers. Suggested growth ^loci (arrows) ^{for the case of} (b) ^{are shown in} (c). ^A growth ^{locus for the case} of (a) ^{is shown}

as an inverted micelle on the top ^of (d). Water is shown

as cross-hatched The curves that do not contact water are to

guide the eye; they represent ^a continuous matrix of hydro-

carbon tails.

available X-ray ^{flux limits our} time resolution on this kind of

experiment

^to

roughly

100 seconds. Within this time resolution, ^the X-ray patterns ^are

fully developed, indicating

^{the domain} exchange

equili-

brium to be at least this fast.

It is

important

^to

recognize

that the 2-dimensional

hexagonal packing

^of

Hn

tubes deduced from the X- ray diffraction of

randomly packed Hn

^{domains is}

consistent with both

straight

^and curved tube bundles.

The literature contains many examples

(e.g. [14])

which demonstrate

Hn

tubes often are curved Com-

mon motifs, ^as abundantly ^{clear in}

figure

7, ^include spiral and toroidal whorls replete with defects where the tubes branch. Note that toroids and

spirals

^with

branch defects are mechanisms

whereby

^{the water-}

containing

^{tube centres are}

kept

^from

hydrophobic

contact : there are no « ends » to worry ^over.

Striking stereoscopic

demonstrations of the toroidal motif in whole, native rod outer segments may be seen in

figures

^{1 and 2 of} [14].

It would be of interest to

analyse

the defect struc- tures internal to the

Hn

domains in terms of develo-

pable

^domains

[15]

and

hexagonal

order, ^{as described}

by Bouligand [16].

However, ^{it is unclear} if, ^for

Fig. ^{6. - The} Lex-Hu equilibrium shifts toward the La phase

a lower temperatures. ^The diffraction from a hydrated ^native

membrane specimen ^{at 61 °C} (a) clearly shows the meridional

( ± 1,0) hexagonal ^{orders and the weaker} off-meridional ( ± 1, + 1) ^and (0, ± 1) orders. If the temperature ^{is sud-} denly dropped ^{to -} 5 °C, ^the hexagonal ^orders disappear (b),

but immediately re-appear upon reheating ^{to 61 °C} (c).

These diffraction patterns ^{are each 5} minute exposures

acquired ^{with an} electro-optical X-ray ^detector [12]. ^The patterns ^were quadrant averaged, displayed ^{on a TV monitor}

and photographed. ^The strong first and second L ^{orders are}

shown highly over-exposed ^{on the} meridian inside the

hexagonal ^order ring. ^{If the} specimen ^is equilibrated against

a higher humidity, ^the temperature, ^{below which the} speci-

men is all Lex, ^rises (data ^not shown).

example, ^the language ^of

developable

domains is

appropriate for these specimens, since the

required geometric

constraints [15] may ^{not be met} (e.g.,

spiral

structures can be seen). ^It would also be desirable to have large

regions

^of pure

HII

^{with a} low defect

density.

Such

regions

^{are not common in the}

micrographs, possibly

because the

specimen

^is

mostly

^lamellar.

The situation _may be different with _pure

lipid speci-

mens. This presents ^an

interesting

^{avenue for future} exploration.

3. Discussion.

The unusual features of the biomembrane

liquid

crystals ^{we have examined are} summarized as follows :

1) ^{The bulk of the}

liquid

crystal ^{is in a}

Lex

^{state with a}

small fraction in the form of

interspersed Hli

^domains.

2) ^The

HII

^{domains are} always

geometrically

align-

ed such that a layer ^of

hexagonal

^{tubes is} co-planar

with the

adjacent

host lamellae.

198

Fig. ^{7. - A} freeze-fracture electron micrograph ^{of a native}

membrane specimen, reproduced ^from [11]. ^The closely spaced striations are parallel Hn ^{tubes. Note} that these often

occur in swirling ^{or toroidal textures. The} fine-grained, planar regions ^{are fractured} bilayers. ^The pebbled planar regions ^{are believed to be} protein aggregates imbedded in the bilayers (see ^{[7, 11} ]). The bar in the _upper left represents 200 nm.

3) ^The Hn layer repeat ^is often observed to be commensurate with the host lamellar repeat.

4) ^The HII ^{domains have} been observed to have external crystal ^faces.

5) ^The relative fraction of the

specimen

^{in the} HI, phase ^{decreases at} high

hydrations

^{and at low} tempe-

ratures. This mass movement is

accomplished

^{on a}

time scale faster than a few minutes and is reversible, including ^the

geometrical coupling

(e.g., co-planar layer lines).

6) ^{On a} scale,

large compared

^{to their} diameter,

the

Hn

^{tubes, curve and wander, often}

exhibiting spiral

and toroidal patterns ^{with numerous}

branching

defects.

7) ^The

HII

phase appears ^to be devoid of _pro- tein [11 ].

There is evidence that the pecularities ^{of these}

liquid

crystals ^{are not confined to the} photoreceptor

protein

system. ^{The most} obvious feature of the

L,,,-Hi,

layer

co-planarity

^{is the} appearance of a

ring

^of X-ray

orders at about 40 A Bragg spacing ^which appear

strongly ^on the meridian and

weakly

^{at + 600 to it.}

Finean et al. [17],

performed

X-ray studies of dried

multilayers

^{from a} variety ^of

biological

^membranes

noted that « One diffraction

ring

^{which occurs at a} Bragg

spacing

^{of about 40 A in most if not all dried}

samples frequently ^shows

pronounced

intensifications at about 300 to the equatorial ^{axis as well as on the}

meridian », This was interpreted ^{as a}

separated lipid

phase ^{which was «}

readily

^modified by

heating

^or

cooling

^{». No further} characterization of the

phase

was

performed.

^The diffraction patterns

displayed

in Finean et al. show off-meridional orders that are

less intense than the on-axis orders. It is straight-

forward to show [I I that if the reflections arise from

co-planar hexagonal domains with a random distri- bution of directions of the tube axes parallel ^{to the} substrate, then the ratio of the

peak

^{off- to on-meri-}

dional axis

intensity

is reduced by ^{the ratio}

where I(a) ^{is the}

intensity

distribution of the meri- dional order and a is the polar angle ⁱⁿ the detector

plane

centred in the forward direction. This would

explain why

the Finean et al. patterns

display

^six-fold

rotational symmetry ^with respect ^to position ^{but not}

with respect ^to

intensity.

The six-fold reflections observed by Finean et al. are often smeared along ^a

ring

^{due to the} high ^mosaic spread ^{of his} specimens.

Consideration of both the Finean et al. [17J ^{and our}

data suggests ^that

geometrically aligned L(.(-Hn

phase

co-existence occurs in a

variety

of dried biomembrane

specimens.

^It is of interest to ask as to the role of _pro- tein in the phase

separation.

Proteins confer

specificity

to

biological

membranes.

Evidently,

^{different mem-}

branes

yield

^similar behaviour; ^this

implies

^that pro- tein

specificity

^{need not} be invoked in

explaining

^the

La-HII

phase separation. ^{A common} attribute of _many membrane proteins ^{is that} they ^are

bulky ampiphiles

which span membranes [18]. ^These proteins ^have polar ^{residues at the ends}

jutting

^{in the} aqueous medium and

hydrophobic

^residues

girding

^the middle, securely

anchoring

^the

protein

^{in the membrane} hydro-

carbon. Relative to the

lipid

chains, ^the proteins ^{are not}

readily

deformed. The currently

accepted

dogma [19]

is that these

proteins

^{are dissolved in a} two-dimensio- nal sea of

lipids.

The melted

lipid

chains fill in the crevices of the sides of the

irregularly shaped proteins.

It is difficult to see how such a

bulky ampiphile

^{can be}

accommodated in the

tight

geometry ^{of the} Hn

phase.

Indeed, ^electron

microscopy,

X-ray diffraction [11],

and infrared and ultra-violet

spectroscopies

[20, 21 ]

all demonstrate

rhodopsin

^{in our}

specimens

^{to be}

largely

intact and confined to the lamellar phase. ^{It is}

likely,

however, ^{that the}

irregularly shaped proteins

favour the retention of

lipid

^{in a lamellar} phase ^{due to}

packing

considerations and the need to

keep hydro- phobic protein

^{surfaces out of} aqueous ^contact.

Although proteins

may ^{serve to} stabilize the

La

phase, ^the

Hn

^domains appear ^to be _pure

lipid

[11 ].

It is known that certain _pure

lipids,

^{such as} PE, ^form

Hn

phases ^{whereas others, such as the}

phosphatidyl-

cholines

(PC),

aggregate ^as

bilayers

under identical