States of phase transitions in biological structures

Progress

in Surface Copyright

sOO79-6816(96)00003-Z

Science. Vol. 51. No. 3, pp. 233-261. 1996 0 1996 Published by Elsevier Science Ltd Printed in Great Britain. All rights resewed. 0079-6816/96 $32.00

STATES OF PHASE TRANSITIONS BIOLOGICAL STRUCTURES M. KRIECHBAUM

IN

and P. LAGGNER

Institute of Biophysics and X-Ray Structure Researchof the Austrian Academy of Sciences,Steyrergasse17, A-8010 Graz, Austria

Abstract The mechanismsand kinetics of phospholipidphase transitions - induced by temperaturejumps or pressurejumps and investigated by time-resolved X-ray diB?action using synchrotron radiation - are discussed.Mechanistic models for these thermotropic and barotropic phase transitionsobtained from thesejump relaxation measurements under non-equilibriumconditionsare discussed.

Contents 1. Introduction 2. Linear

or Non-Linear

3. Phospholipid 4. Diffraction

Non-Equilibrium

Phases and Transitions from partly

ordered

and disordered

lipid,systems

A. Lipid polymorphism 5. X-ray

diffraction

with synchrotron

radiation

A. Time-resolvedX-ray dif%actionon lipid phasetransitions:T-jumps B. Presurejump induced phasetransitions C. Radiation damage 6. Conclusion Acknowledgements References

Acronyms CCD DHPE DMPC DOPE DPPC

Charge-coupleddevice Dihexadecyl-PE Dimyristoyl-PC Dioleoyl-PE Dipahnitoyl-PC 233

234

M. Kriechbaum

ESR rR NSLS PC PE POPE SAXS SOPE W WAXS

and P. Laggner

Electron spinresonance Inf?a-red Nuclear magneticresonance National Synchrotron Light Source Phospahtidylcholine Phosphatidylethanolamine 1-pahnitoyl-2-oleoyl-PE SmallangleX-ray scattering 1-stearoyl-2-oleoyl-PE Ultra-violet Wide angleX-ray scattering

1. Introduction Most, if not, all the existing data on macromolecularstructures in terms of the threedimensionalatomic resolution originates f3om difhaction methodsusing X-rays or neutrons, which are inherently extremely fast as they are based on elastic elementary wave-particle interactions. In reality, however, this speedcannot be used, sincethe diffraction cross-sections are smalland the photon or neutron flux Corn conventionalsourcesis very weak. In practice this means,that the collection of data with high precision and spatial resolution by conventional means,e. g., from a protein crystal, or from a liquid crystallinelipid sample,is a matter of many minutes,at best, or hours. Molecular dynamics, as for instancethe direct cinematographicmonitoring of structural changesassociatedwith biochemical function, has been out of reach of &action

methods.

Information related to theseaspectswas therefore the domainof spectroscopicmethods,NMR, ESR or fluorescence,but their structural information content is comparatively low and restricted to the immediateatomic neighborhoodof the particular probe or nucleusused.The aim still has to be, therefore, to obtain a motion picture of an entire functional, macro- or supramolecular entity during its specific action. Membrane transport is one of the most attractive objects for this approach. The present situation,with synchrotron radiation sourcesexisting in manyplacesaroundthe world, where Xray fluxes many orders of magnitudeshigher than Corn conventional laboratoy generatorsare available, gives rise to optimism. In this article, the most recent advancesin this field are reviewed, showingthat the millisecondtime-resolutionis now feasiblein X-ray diffraction, so that the gap betweenstructural and functional information may be filled in molecularbiophysics.

States of Phase Transitions

2. Linear or Non-Linear

in Biological

Structures

235

Non-Equilibrium

III enzymology, also in membranetransport, the steady-stateapproachhas often been usedto get to terms with overall and partial reaction kinetics.However, no detailedinformation on the existenceor lifetimes of intermediatestatesis availablesincethe intermediatestaiesof the transition are not sufEciently populated by this approach. Hence, this type of kinetic studies answersthe questionof how fast a biochemicalreaction may proceed andwhich type of rate-law it follows under a linear set of conditions, but leavesthe dynamicsof molecularmechanisms largely untouched. This is essentiallya stationary approach close to equilibrium formalisms: thermodynamically, a linear relationship between driving force and fluxes (or affinities and reaction rates)prevails. This linearity of reaction laws, however, appliesonly to smalllimitsnear an equilibrium.At larger deviations Corn equilibrium this linearity is lost and qualitative changesoccur. Hence it is impossibleto extrapolate Corn the linear to the non-lineardomain,where entirely new structures or processesmay be developed, as a consequenceof fluctuations in time and space [l]. Returning to the specific situation in membranetransport, however, all ingredients for this concept, as self-organization, domain structures, fluctuations, switching processesand large gradients, are present, so that one should by no meansneglect the concept of non-linear behavior, even if direct evidenceseemsstill lacking: it may only be due to the fact, that we have so far not been able to enter experimentallythe appropriatetime and spacescales,Here, again, the methods of time-resolved X-ray dtiaction

with synchrotron radiation give reason for

optimism.

3. Phospholipid Phases and Transitions The thermotropic, barotropic and lyotropic mesomorphismof phospholipidsas major constituents of biological membraneshas for a long time stimulated interest in biophysical research,and it is generally assumedthat certain membrane-associated actionsin the living cell, such asfusion, pore formation and possibly alsothe regulation of enzyme activity, may invoke transientchangesin lipid structure similarto those observedin pure lipid systems(Fig. 1). Yet, a detailed understandingof the molecular mechanisms and the dynamic processeswhich govern these transitions is still lacking, and this is also one reasonfor the rather speculative roles attributed to lipid mesomorphismin biological membranesystems. Apart Corn this aspect of biological functionality, the topological and mechanistic interrelationshipsbetween different ordered lattices, as they are statically well describedfor

236

M. Kriechbaum A. CLASSES

and P. Laggner

OF POLYMORPHIC

Lamellar

6. SOME FORMS

LIPID PHASES

Hexagonal

OF LOW-TEMPERATURE

Cubic

LAMELIAR

PHASES

Cfystalline

Gel

Ripple

Phase

P

P

Fig. 1. Some of the characteristic lipid phase structures in schematic representation. A: The more frequent types of polymorphic phases;note the differencesin local curvature at the lipid-water interface. B: The most frequent types of bilayer structures in lamellar lipid phasesbelow the chain-melting transition.

States of Phase Transitions

in Biological

Structures

237

many synthetic phospholipid species,pose interesting problems of more general, physicochemicalnature in correction with theories of phasetransitions.One of the central questionin this context relates to the occurrence and mechanisticsignificanceof intermediate structures which may be formed transiently during the process.

4. Diffraction

from Partly

Ordered

or Disordered

Lipid-Systenis

It is a widespreadbut mistakenbelief amongbiologists,who are most familiar with the spectacularsuccessof protein crystallography, that X-ray diEaction is limited to the availability of well-developed single crystals. In actual fact, with biological membranes, the main contribution by X-ray diEaction was madethrough its potential to obtain structural information also from random or partly ordered systems.Figure 2 showsa schematicrepresentationof the nature of structural information that can be extracted from such studies on lipid model membranes.This information can be directly juxtaposed to the thermodynamicdata defining the relative stabilities and transition enthalpies as obtained e.g. from differential scanning calorimetry. Recently, Chung and CafEey [2] have reported on a combined experimental approachto obtain X-ray structural and calorimetric data simultaneously,which is certainly of high value in directly assigningcertain structural changesto the respectivetransition enthalpies. In the following sections, a brief recollection is given on the various approachesby Baction

techniquesto study problemsrelevant to supramolecularlipid structures.For more in-

depth information the reader is referred to comprehensivemonographsand reviews on the generaltheory of X-ray diEaction (e.g. [3-S] andX-ray small-anglescattering[6-91). As will be shown, X-ray difI?action offers a highly convenient and informative way to study lipid polymorphism by a comparatively simpleapproachwhich does, in most cases,not require extensive procedures for data analysis. In this field, X-ray methods have become comparablein their easeof application to spectoscopicor thermodynamictechniques,and thus form an integrating componentin the methodicalarsenalof membranebiophysics. A. Lipid polymorphism Polar lipids, such as phospholipids,when dispersedin water, form aggregateswith a certain type of local symmetry, that can be classifiedcrudely in lamellar, hexagonal, or cubic structures,dependingon the nature of the lipid and the physico-chemicalstate of the system(T, p, concentration, and nature of saltsor other additives). Thesehydrated lipid structuresdisplay, to good approximation, the characteristicsof phasesin the thermodynamicalsense,i.e. they

238

M. Kriechbaum Multilarnellar Liposomes

and P. Laggner Accessible Information

What X-Rays “see*

__+

o Bilayer Thickness

-

o Waterlayer Thickness

___)

o Surface Area I Lipid

+

o Electron Density Profile

+

o Chain Packing Geometry

Unilamellar Vesicles 4

o Bilayer Thickness

d

o Surface Area / Lipid

*

o Mass / Unit Area

+

o Electron Density Profile

Resolution: -10.1000A

Fig. 2. Scheme of the iufon&tion available Corn X-ray &action on lipid/water dispersions in the partly ordered form of multilamellar liposomes and in the form of unilamellar vesicles.

States of Phase Transitions

in Biological

239

Structures

follow Gibbs’phaserule and exhibit phasetransitions.With the exception of cubic phases,which can grow to large monocrystallinedimensionsof the size of the samplecontainer, thesephases normally are present in microscopic crystalline domains, randomly oriented throughout the sample,sothat the x-ray beam(its crosssectionbeingmuch larger than the individual crystallite) “sees”the situation of a crystalline powder. This has the effect, that all lattice planesmeet the Bragg-condition simultaneouslythus leadingto a powder-pattern of concentric rings about the direction of the direction of the primary X-ray beam A schemeof this powder diEaction is shownin Fig. 3. If availableto suflicient resolution, the powder difhaction pattern provides all necessary information to define the crystallographicunit cell symmetryand dimensions.In the generalcase of three-dimensionalcrystals the indexing of a large number of individual reflections can be an elaborate procedure. With phospholipid dispersions,except for complex cubic structures however, the problem is grossly simplifiedbecausenormally only relatively few reflections are sufEcient to describe the type of symmetry (e.g. lamellar or hexagonal) and the relevant dimensions.This is due to their liquid-crystallinenature, i.e. the fact, that a structural regularity is only expressedat the supramolecularlevel, while the individual moleculesand atomic groups are not fixed in a long-rangeordered crystal. Owing to the fhct that the supramolecularlattices of polymorphic phases can have characteristicdistancesin the order of 10 to several 100 A, and the recipocity between realspacedistancesd and scatteringangle28 asexpressedby Bragg’sequation, n.1 = 2dsint3

(4.1)

the correspondingdiffraction signals(with wavelengthsh in the order of 1 A) lie in the smallangleregion, typically between somelo- 100mrad. There is, however, also important structural information to be extracted from the wideangle region, correspondingto the dimensionsof the subcell-latticesof the hydrocarbon chain packing if they are not in a liquid disorderedstate. Ideally, thesetwo regionsof the di5action pattern, which pose quite different demandson the detection system(4.ib), shouldbe measured simultaneouslyto characterize the structure of a given phase [lo]. The most frequently encounteredmesomorphicphasesare describedbelow with respect to their typical diffraction features. (i) LamelIar

phases.

(ia) Small-angle

diffraction.

Depending

on the chemicalnature of the lipid, the state variables

(T, p, and degree of hydration), or the presenceof co-solutes,the lipid bilayer can occur in

240

M. Kriechbaum and P. Laggner (4

nA=

2 d sin0

Deleclor

Fig. 3. (a) Schematic representation of &action of a wave on a lattice of planes (Bragg diffraction); (b) the optical arrangement in a powder diffraction experiment; the insert shows an idealized diEaction pattern, i.e. the radial intensity profile through the di5kactogram at the detector.

States of Phase Transitions

in Biological

241

Structures

various structural forms, such as normal or interdigitated, or partially interdigitated, which all canbe characterizedby their respective small-anglediEaction. In this case,the existenceof only integral multiples of the position of the &$-order reflection indicatesa onedimensionallattice. The structural regularity relates only to the periodic repeat of the lipid bilayer together with the water of hydration. The repeat distanced can be simply calculatedl?omthe angularpositionssn of the reflection peaks,accordingto d = n/s, = h/(2si&,)

(4.2)

If the volume fraction of water f, in this phase(not in the total samplevolume) is known, e.g. from a seriesof measurementsof d as a function of water content, the thicknessof the lipid bilayer dl can be calculatedCorn dl= d.( l-f,)

(4.3) In addition to these integral structural parameters,the powder diBaction patterns can be analyzedalso in terms of the electron density profile, p(x), centrosymmetricaland normal to the bilayer plane. This requires the measurementof the intensities I(n) (with n...order of the difbaction peaks) and the determination of the signs of the amplitudes+41(n). The relation betweenp(x) and I(n) is given by p(x) = (2/d)*C ?&I(n) cos(2xxn/d)

(4.4)

Severalproceduresfor the determinationof the signsin this equationhave beendescribedin the literature (for reviews, see[ 12, 131).Particularly elegantways ofveriljing the choice of signsfor the amplitudesare given in neutron scattering by contrast variation with H2O/D20-mixtures l?31. (ib) Wide-angle diffraction.

At temperatures below the chain-melting transition, the

hydrocarbon chainsof membranelipids adopt ordered states,in which the motions in the plane of the bilayer are frozen. This may occur in discrete stages:Iirst, only the lateral motions are inhibited while the rotation about the long molecular axis is still fast. This can lead to a hexagonalor pseudohexagonal(2-D rectangular) packing of the chain rotation cylinders, which have a specific signature in the wide-angle diEaction patterns (Fig. 4). At even lower temperatures,the chainsmay adopt a crystalline configuration, with a more differentiated wideangle diffraction pattern. Recent examples,where both small- and wide-angle dilEaction were usedto analyselipid polymorphismcan be found in [ 141,for diehexadecyl-phosphatidylcholine, in [ 151for diacylglycerols, and in [ 161for glycolipids.

M. Kriechbaum

242

and P. Laggner 1

I

I

a. Liquid

chains

4.5

1

ii

A

b. Nonspecific

chain

4.2

i

4.2

i

packing

hexagonal,, a = b = 4.2

A A

A 4.1

pseudohexagonal (2-D

rectangular) a >*

b

4.10

c. Specific

triclinic

tight

subcell

chain

packing

#I-. @a 8t-t-i tt tI 888 1 0.15

4.16

ii

i

ii

4.24

ii

4.42

ii

3.92

ii

n I

I

1

0.20

0.25

0.30

s

(ii-y

Fig. 4: Left: typical examplesof hydrocarbon chainpacking symmetriesviewed in projection onto the bilayer plane; right: the corresponding X-ray wide-angle diE?actionpatterns. From Small, [67], with permission.

States of Phase Transitions (ii) Hexagonal

in Biological

Structures

243

phases. Closely analogousto the one-dimensional approachoutlined above for

lame&r phasesis the analysisof powder di5action patterns from two-dimensionalhexagonal lipid phases.The fingerprint of a hexagonal phasein powder patterns, which in fact has been proven to be more critical than the 3 lP-NMR signalis the sequenceof spacings s = (1,‘/3,2,47,3,,,)/d.

(4.5)

The first d-spacing(correspondingto the [ l,O] Bragg peak) is related to the distance a between the water cylindersby a=2dfd3

(4.6)

(seealsoFig. 5). Again, this distancecan be geometricallyseparatedinto the lipid layer thickness and the water cylinder radiusby a volume fiaction calculationsimilarto the one indicated above for the lamellar case. Similarly, the molecular surface area at the lipid/water interface can be calculated. All equations are listed and discussedin [17]. Methods to reconstruct the 2-D electron density relief of the hexagonal HII phasein the planevertical to the cylinder axes corn the observed X-ray intensities have been recently refined and extended to electron-density reconstructionof a non-circular averagedshapeof the water-tubes[ 18, 191.

Fig. 5. Geometric schemeand molecularpackingin the hexagonalHII-phase.

244

M. Kriechbaum

and P. Laggner

(iii) Cubic phases.In the context with membranefusion and, more generally, with the question of membranecurvature, the cubic phasesof lipids have recently attacted increasinginterest. Such three-dimensionalperiodic structures have been first observed in bacterial lipid extracts (Pseudomonasfluorescerq [20]). Various crystallographic spacegroups for cubic phasesof membranelipids have beenfound and discussed by X-ray difbaction analysis[21-261. In all cases so far, these structures have been found to lie in between the L, and HII phases,and it has therefore been postulated that cubic structures play a general mechanistic role in the interconversionbetweenlamellarandhexagonalphases[27, 281. From the practical point of X-ray maction,

both the measurementand the analysisof

cubic phasesis definitely more di&ult than that of lamellaror hexagonalones.Cubic phasesof membranelipids tend to form macrodomainsin the samplesandtherefore the powder difbaction approachis often unsuccessfulin picking up the characteristicreflections. They are also often metastable,their generation can be enhancedby temperature cycling, and in general their formation is poorly reproducible [26]; moreover, the pattern of discrete reflections may be blurred by a strong diise scatteringbackground, so that an insufftcientnumberof peakscan be indexedand assignedunambiguosly.For reliableresultsit is therefore essentialto have a precise record of the samplehistory and its effects on the observeddiffraction pattern. Preferably twodimensionaldetectors (lihn or 2-D position sensitive detectors), as well as samplerotation shouldbe employed.

5. X-ray

Diffraction

with Synchrotron

Radiation

Synchrotron radiation from electron or positron storage rings [29, 301 offers several excellent features for performing X-ray diffraction studies,such as extremely high X-ray flux, spectral tunability from “hard” X-rays to “soft” UV range (dependingon the energiesof the acceleratedparticles), linear polarization, and pulsedtime structure. The high X-ray flux in the order of lOI photons/s/mm*- gain factors of about lo5 as comparedto conventional laboratory x-ray sourcescan presently be obtained - has led to new and exciting applicationsfor timeresolvedX-ray tiaction,

also in the field of membranestructural studies(for reviews, see 12,

3 I-351). Especiallytime-resolvedX-ray diBaction experimentsrequire the highestpossibleflux on the sampleand fast detectors combinedwith fast data acquisitionhard- and software, capableto measureand store tiaction

patterns in millisecondtime resolution.A high optical performance

States of Phase Transitions

in Biological

24s

Structures

is achieved by combinationsof X-ray focusing and/or mono-chromatizing elements.Figure 6 showsas an examplethe designof a new modemSAXS beamlineto be built at the synchrotron ELETTRA, Trieste, Italy, which is optimized for these purposes [36]. As X-ray detection systems1-D or 2-D detectors can be employed. Suitable 1-D detectors are gas proportional countersbasedon delay line or chargedivision technique 1371,2-D area detectors can be either multi-wire chambers,CCD or image-platedetectors[38]. A detailedoverview and discussionon beamline optics and detector systemsat synchrotron sourcesis given by Helliwell[39]. Detector

Asymmetric

Cut

Beam Defining

Slit-

Fig. 6. Sketch of the small-angleX-ray beamlineto be built at the synchrotron ELETTRA, Trieste, Italy. It showsthe optical designwith the monochromatizing elemets (two asymmetric cut crystals), focussing elements(segmented toroidal mirror) and the beam-sizeconfining elements(slits) betweenwiggler and detector. At this point, a brief technical remark is necessaryto illustrate the focal points of the present developmentin time-resolvedX-ray diffraction. The possibility openedby synchrotron radiation sourcesto take millisecondexposuresis but one prerequisitefor cinematographicX-ray studies. Another oflen neglected point concernsthe timing and triggering of the phasetransitions. In most of the studies so far, temperature has been chosen as the variable for triggering the transitions.Originally, simply external heatingby thermostatfhrid [3 l] or heating by a heat gun [40] have been applied. With such methods,however, the problemsof slow heat conduction

246

M. Kriechbaum

and P. Laggner

within the aqueous samplespose severe limits: the inevitably occuring internal temperature gradientslead to the fact, that at any instantthe X-ray beam“sees”a broad distribution of states. This limitation cannotbe overcome: the faster the external heating,the larger will be the internal gradient, and consequently no clearcut structural information can be drawn from such experiments, unless the structural processesare much slower than the time-scale of Tequilibrationwithin the sample,in which casethe use of synchrotronradiation is obsolete.What is really required are defined jumps amplitudes in the thermodynamic variables (T, p), concentration of an additive or an external appliedfield (hydrodynamic, electrostatic, magnetic) so that the entire system under investigation crossesthe thermodynamic phase boundary instantaneously. A. Time-resolved

X-ray

diffraction

on lipid phase transitions:

T-jumps

In the searchfor better methodsto inducetransitionsrapidly, it becameclear that radiative heating through direct absorption within the sampleshould be by far superior comparedto conductive heating. Of the two principal possibilities,microwaves [41, 421, where jump amlitudesof up to 29”Ck. were achieved,or in6ared laser, we have opted for the latter [43-451 due to its optical convenience.The basic idea is to exposethe sampleto a short inf?ared-laser pulse and at the sametime to start time-resolved data collection of the tiactograms.

The

experimentaldesignprinciplesare shown in Fig. 7. Recently, this type of experimentshasbeen extended to simultaneousmeasurementsof the small- and wide-angle region in the reciprocal spaceby employingtwo separateposition sensitivedetectors [46,47]. An Erbium-glasslaser as describedpreviously for T-jump experimentson musclefibres, operating in single-pulsemode, was used asthe heat source [48]. The wavelength of this laser liesat 1.5 pm. At this wavelength water hasan absorptioncoefficient of 6.5 cm-l. A singlelaser puke of 2 ms(FWHM) duration deliversan energy of l-2 J. The laserbeamwas guidedvia two glassmirrors onto the sample,where it covered a cross section of approx. 3 x 6 mm2. The sampleswere contained in a cylindrical glasscapillary in a thermoelectrically controlled steel cuvette holder. The capillary was alignedin the cross-pointposition of the horizontal X-ray and vertical laser beamsby meansof a dummy He-Ne laser. The illuminated samplevolume was approx. 5 ~1.To improve the *ared

absorptionyield and to reducethermal gradientsalongthe

absorptionthickness, a gold-coated glassmirror was placed underneaththe sampleso that the laserbeampassesthe sampletwice on one shot. With optimal tuning, this arrangementproduced T-jumps of approximately 10°C in the sample. Since no s&iciently fast, non-perturbing

States of Phase Transitions

in Biological

247

Structures

temperature probe exists for direct measurement,the magnitudeof the jumps was determined through internal calibration with lipids by lowering the pre-jump temperatureto the limit where the the T-jump produced a phasetransition of lmown T, detectableby the time-resolvedX-ray difbaction pattern. Detailed computer-simulationsof the spatialand temporal temperatureprofiles within the sample(for details of the calculation, see[45]) have shownthat the initial temperaturegradient between laser- and mirror-facing sidesof the sampleis lessthan 2°C and decreaseswith time. The time-evolution of this profile is dominatedby the axial thermodifikion into the unheated regions of the cylindrical capillary, while radial effects and convection into the cooler environment outside the glasswalls play a minor role. The changesin the temperatureprofiles are negligiblein the millisecondto secondtime range, Synchrotron

Radiation Bent Crystal Monochromator

(Ge)

Thermoelectric

Segmented

Mirror

Bent Linear /Sensitive

Position Detector

Fig. 7. Schematic view of the millisecond time-resoled X-ray diiTraction with synchrotron radiation and &laser temperature jump setup at the synchrotron DESY, Hamburg, Germany. In the following, a summaryover the hitherto performed time-resolved x-ray aaction studiesby &laser T-jump shall be given. In the discussionof structural phasetransitionswe have found it convenientto introduce an operationalclassificationbasedon the geometricnature of the long-range rearrangementsinvolved [49]. Thus we use the term homologous for transitions between afEne lattices, i.e. where only the lattice parameterschange, while the

248

M. Kriechbaum and P. Laggner symmetry type remains unchanged (e.g. lamellar-lame&u).

Heterologous

transitions,

on the

other hand, are those for which the symmetry type changes (e.g. lamelhu-hexagonal). Despite its simplicity and lack of theoretical foundation, it will be seen that this system allows for certain predictions regarding the transition mechanism (i) Homologous

transitions:

The LJ - L, transition.

Ethanolamine phospholipids (PE’s) show

a transition from the lamellar-Lp, with all-trans hydrocarbon chains oriented perpendicularly to the bilayer plane, to the lamellar-L, phase with fluid chains. The two lamellar lattices differ only in their repeat period, such that the L&attice

period is by several &units

smaller than that of

LP

ms

40

*

30

1 ‘3; E 20

-

a, E .--c-r

10 l

0

laser power

0.014

0.018

Fig. 8: Time-resohredX-ray diffraction pattern of the Lp - La phasetransition of POPE induced by an IR-laser pulse. The intensity contour line plot for the first 50 msis shownleft. For the first 5 ms each singleframe with a time-resohrtionof 0.35 msis shown(middle) andthe laserpulseprofile (right).

States of Phase Transitions

in Biological

249

Structures

A representativeresult of a temperature-jumpexperimentwith such a transition is shown in Fig. 8. It is evident Corn the rapid decay and formation of the &t-order powder-diffraction signalsof the respective lattices, that the transformationproceedsat the sametime-scaleas the duration of the laser pulse, i.e., less than two milliseconds.There is no sign of intermediate disorder aswould be evidencedby a broad, continuousbackground, and as would be expected for an order-disorder-order mechanism(Fig. 9a). In this particular experiment, the individual time framesfor tiactograms were 350 ps, i.e. aboutten timesshorter than the laserpulse.This artiIicia1time-resolution was chosento demonstratethat the methodhas the potential to enter even the sub-millisecondtime scale, if the T-jump could be shortened.Presently these are the fastesttime-resohtions ever obtainedin Faction

experimentson membranesystems.

Noting that this transition proceedsextremely fast and appearsas a two-state processit is obvious that a martensiticmechanismmay underlie.Martensitic transitionsimply the existenceof strain-free lattice-equivalent transition @ties that move rapidly throughout the system A plane is lattice-equivalent ifit combinesthe locations of both the initial and Iinal lattice positions. It is important to note, that the particular geometricrelationshipsbetweenthe initial and Iinal lattices require large-scaledeformations.

TRANSITION

DISORDERED

TRANSITION

ZONES

LATTICE

PLANE

A

\ LATTICE

A

LATTICE

B

_---

---_________-

a

1Dl ~:--Fr -..* --._ -. y-q. ZONES

OF DISORDER/LOSS

OF

COHERENCE

MINIMAL

LOSS

OF

ORDER

6 COHERENCE

Fig. 9. Schemeof two alternative transition mechanismsin lamellarphases. In an attempt to rationalize these findings, in particular the notion of coexistenceof the two lamellar structures with different repeat distanceswithout intermediatedisorder, we have postulated [44] that the transition occurs by cooperative folding about a disclinationplane, as indicated in Fig. 9b, which propagatesrapidly through the system The ratio of the repeat

250

M. Kriechbaum

and P. Laggner

distancesis simply related to the cosineof the disclinationangle. This mechanismaccountsfor the conservationin lattice order andfor a minimumin defect formation. Transitionsof this type are known to metallurgistsasMartensitic “Umklapp”-transitions[50] (ii) Heterologous transitions: Structural intermediates. In these transitions, the lattice dimensionsand the symmetry types are changed. The best studied casesare the lamellar to inverted-hexagonal (L,-HII)

and the lamellar-gel-to-ripple-phase (L~PJ)

transitions,

respectively. In both cases,the temperaturejumps have shown the existence of short-lived intermediates. (iia) The lameliar-to-inverse hexagonal (HI,) transition. Ethanolamine phospholipidsas discussedbefore show a secondtransition L-H,, at temperaturesabove the main chain melting transition. This involves a major changein symmetry, i.e. from lamellarbilayers to hexagonally packed tubes (Fig. 5). From slow-scanningtimeresolved X-ray *action

data it had been

concluded, that this is a two-state process,with extensive coexistencebetween the initial and final phases[ 12, 5 11. The analysisof fast T-jump X-ray difhaction data (Fig. 10) provides additionalinformation on rates and mechanisms.Thesecan be summarizedas follows: As in the previous caseof the pretransitionthe relaxation showsmore than one kinetic component.The initial relaxation step, occuring rapidly in times of lessthan 5 ms, involves a shrinkageof the Lo-structure by about 4 A-units or 7%. After a lag-period of approximately20 ms,the first signsof the hexagonallattice becomevisible, with the first-order reflection initially at spacingssmallerthan that of the final H~j+tructure. This reflection grows in intensity at the expenseof the coexisting ~.,hh-signal, and gradually shifts to its final equilibriumposition within about 10 seconds.In the time-span availableto this type of experiment,i.e. lessthan 10 s, the transition is not complete.This agrees with the slow-scanresults,where the limiting transitiontime was found to be about 12 s [ 121. A structural model mechanismwhich accounts for these observationsis schematically shownin Fig. 10. Again the rapid initial transformationto the thinner lamellarphaseis likely to be best describedby a martensitictype, with a dischnationat a plane by an angle of 19” (note that 51/55 = cos 19”). This transition planemoves rapidly through the structure. Nucleation of the first tubular structures is facilitated by the close approach of adjacent bilayers, and it is thought, that this again happensat a second,lattice-equivalenttransition plane, which however,

States of Phase Transitions

in Biological

Structures

laser pulse II I

! I 10

50100

5mlooo

time (ms)

-b

rapid

-

slow plane movement

Fig. 10. Time-resolveddevelopmentof an IR-laser pulse induced & - HI~ phase transition of SOPE. X-ray *action patterns (top), d-spacings(middle) as a fkwtion of time within one 1 s after the pulse(time-resolution 1 ms) and schematic model(bottom).

251

252

M. Kriechbaum

and P. Laggner

moves more slowly. The initially distorted hexagonallattice annealsin times of secondsto the Iinal equilibriumstructure of the HII - phase. This model accountsfor severalgeneralfeatures of the transition: First, the formation of the thin lamellar structure is the coordinated step to mutual approach, so that adjacent monolayerscan comeinto firsional contact. Second,it provides a rationale for the fact, that the tubular structuresare approximately but not quite coplanarwith the initial lamellae.Again, it has to be noted that this mechanismappliesstrictly only to the non-linearnonequilibriumsituation, whereas under equiliirium conditions the formation of the thin lamellar phase cannot be observed.There, localized tumor-relatedfluctuationsin the interbilayer distancemust provide the approach of apposingmonolayersto get into Iirsional contact, which in turn may trigger the transformationof larger domains.In any case,however, there is no evidencefor the existenceof intermediatestructuresof other symmetrythan lamellaror hexagonal,ase.g., micelles.Finally, it can be stated, that the process of transformation under non-equilibrium conditions in this synthetic model systemoccurs rapidly enoughto be relevant to biological membrane-connected phenomenasuchasfusion or local pore formation at the millisecondtime-scale. (iib) The pretransition:

Dissipative intermediate structures. In contrast to the above

situation of PE’s, phosphatidylcholines(PC’s) with two identical, saturatedC-14 to C-18 acyl chains,do not directly transform corn the LJ (in this caseLp), with the chainstilted (by an angle of about 30 degreesto the bilayer normal) to the L, phase,but show a stable“ripple” phase,Pp betweenthem (seeFig. 1). The lateral chainpacking in the averageplane of the rippled layersis of hexagonal symmetry. Despite many theoretical approaches,the origin and physico-chemical nature of this phaseis not yet fully understood.The transition from the lamellargel LFphase to this ripple phaseis calledthe pretrumition, sinceit occws severaldegreesbelow the main chain melting transition. Its enthalpy andvolume changesare only a fraction of the vahresfor the main transition. Typical time-resolved small-anglepowder difl?action scansof the pretransition in DPPC are shownin Fig. 11. They show at leastthree kinetic components:In an initial, fast stepwhich occurs instantaneouslyat the millisecondtime scale,the appearanceof a peak at the high-s side of the first-order reflection of the initial Lp.-phasesignalizesthe formation of a new lamellar lattice of narrower spacing(L*). For variable lengthsof time, dependingupon the temperature reachedafter the T-jumps, thesetwo lattices coexist. The relative amountsof the two lattices, inferred corn the peak intensitiesalsodependon the temperature,suchthat at higher relaxation

253

States of Phase Transitions in Biological Structures

N

Fig. 11. Time-resolved small-angle difEactograms DPPC/water after a laser-induced T-jump.

of the pretramition

of

254

M. Kriechbaum

and P. Laggner

Intermediate Free

State chain

rotation


22s ppS (ripple

phase)

Fig. 12: Structural model for the mechanismof the DPPC-pretransitionunder Tjump conditions.The first, rapid stepis the formation of a thin (58 A) intermediate lattice, by a martensitic “umklapp” mechanism.Coexistenceof many such zones with the parent lattice leadsto a transient ikstration, which lastsfor several 100 rns, and fkally annealsslowly in times of minutesto hours into the equilibrium ripple phase.

States of Phase Transitions

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temperaturesthe narrower lattice predominates.Thesetwo lattices mergein times of 10 ms to 1 s to form a single,broader peak closein position to the original Lyspacing. In a third step, with half-lives between 0.5 and 3 s, this peak transformsinto an asymmetricpattern, which is similar to that seenunder isothermalconditionsin the Ppphase. A tentative mechanisticmodel for this processis shown in Fig. 12. The present kinetic analysisis limited to maximally 10 s, and therefore any subsequent,slower annealingprocesses,for which there exists someexperimental evidence[52], are beyond our considerationhere. Finally, it is of interest to compare this transition mechanismwith the situation as it appearsunder near-equilibrium conditions, i.e. long incubation at temperatures within the transition range, or by very slow heating [53, 541. The X-ray small-anglepatterns obtained in this mode show the progressivelossof long-rangeorder as deducedfrom the absenceof sharp reflections.No trace of L* structure canbe seen.It is evident therefore, that the pathways of the transition differ qualitatively between,the near-equilibriumand the extreme non-equilibrium cases.Thus it appears,that under non-linear nonequilibrium conditions the system is driven through an ordered, dissipative structure by the large free-energy diierence between the unstablestatejust after the T-jump and the final equilibriumstate. B. Pressure-jump

induced

phase transitions

In principle the samephase transitions as discussedin the previous chapters, can be induced by hydrostatic pressurechanges.As an examplethe simplified p-T phase diagramof DOPE in excesswater is shownin Fig. 13 (So [55]). To study the dynamicsand kinetics of these transitions,p-jump techniques- analogousto T-jumps - in combinationwith time-resolvedX-ray difbaction have beenintroduced. Sincethesearevery recent experiments,only a short overview is given. Although the instrumentaldesignand setupfor a p-jump apparatusand a high pressureXray cell is technically more complicatedthan a laserT-jump setup, it has someadvantages:fast hydrostatic p-changes- and hence also phasetransitions - can be made in both directions (pressurizationand depressurisation jumps), the pressureis transmitteduniformly in the sample (no p-gradients), any initial and final pressurebefore and after a p-jump can be controlled and held constant (within the physical limit of the setup). Thus, the p-jump amplitudecan be easily varied which allows detailed kinetic studies. Additionally, p-jumps can be made at different temperaturesand a detailedexploration in the p-T phasediagramis possible.As a drawback,the simultaneousadiabaticT-changeassociatedwith the p-jump, canbe considered.

M. Kriechbaum

256

and P. Laggner

Specifically, p-jump experimentsare performed by quickly openinga vale between a the samplecell, kept at a certain initial pressureandan external reservoir, kept at a different pressure level, thus increasingor decreasingthe hydrostatic pressureto its final value within a few ms. Currently the upper pressurelimit for thesecells,either madeentirely of Be-metal [56, 571,or of stainlesssteel with Be-windows for the X-rays [58, 593 is about 1.8 kbar and 3 kbar, respectively. Mencke et al., [55] used a repetitive technique involving oscillating pressure changes,which has great potential in gaining&rther insight into the kinetics and mechanismof lipid phasetransitions[60].

-20

I0.0

0.5

Pressure

1.0

1.5

2.0

(kbar)

Fig. 13: F-T-phase diagramof DOPE in excesswater. The morphology includestwo lamellar phases,LJ and L,, respectively, and the Hlr - phase.Measurementshave shown the existence of additional phasesat high pressurein the LJ - phaseregime

1551.

States of Phase Transitions

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257

Structures

So far, the barotropic Lp - Lo phasetransition of DHPE [61] andPJ - L phasetransition of DMPC [62] have beeninvestigatedby time-resolvedX-ray difliaction, applying p-jumpsof up to 120bar within 2.5 s. In a more recent work [63] found the intrinsic transit time of the p-jump inducedL, - Lp transition of DHPE at 78“C to be <5 ms, wherasthe depressurizationinduced LJ, - L, transition was found to be slower (1 s). Analyzing the simultaneouslyrecorded timeresolvedSAXS and WAXS difbaction data of this transition, they proposeda mechanism,where the compressedLJ, state of DHPE (124 Mpa) converts first into an umcompressedLJ, state (fast) andthen to the GnalL, state(slow) at 13 MPa. Recently, experimentswith a high pressureX-ray cell attachedto p-jump devices capable of producing p-changesof 1.6 kbar with a rise time of 5 ms [56] and 2kbar within 10 ms [64], respectively, were performed. In the latter work, the LJ - L, and L, - HI~ phasetransitionsof DOPE with a time resolution of 9 mswere investigatedat NSLS Brookhaven, USA, usingfast CCD X-ray detectors. Thereby it wasalsofound that the transition of Lp - L, aswell asL, - LJ proceedsvery fast - like in the casewith T-jumps - and also the p-jump induced L, - HI, transition behavesin the sameway as seenin T-jumps: rapid initial &inking of the L, lattice followed by a slower coexisting growth of the HI, phaseanddecay of the L, phase.The reverse transitionHI, - L, occurs almostinstantaneouslywhen using largejump amplitudes(>lOOObar). It was also observed that the transition rate dependedstrongly on the appliedjump-amplitude, i.e. slow transitions times with smallp-jump amplitudes.As a conclusion, as well as in [61], it wasproposedthat the transport and redistributionof water into and in the new equilibriumphase might be the rate limiting step. Osterberget al. [58] found in p-jump initiated time-resolvedXray difl?ation sudiesof the hydration of the HII phasein DOPE that the hydration followed a power-law kinetics, i.e. non-exponentialrelaxation of the swellingof the HI~ -unit cell spacingof with time in responseto a perturbation by a p-jump. C. Radiation damage The periods of exposureto the intenseX-ray beamfrom the synchrotron radiation source shouldbe kept to a minimumby making the necessaryoptical adjustmentsin the cameraunder the attenuatedX-ray beam (intensity comparableto a conventional, sealedX-ray tube) and by

258

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and P. Laggner

opening the shutter for full brightness only during the time-resolved measurements,i. e., generally lessthan 10 s. Therefore, the total radiation dosisreceived by the samplesis of the sameorder asin experimentswith conventionalX-ray cameras,but over greatly reducedperiods of time. Since any radical-induced secondaryreactions are primarily difllrsion limited, X-ray radiation damageis not a seriousproblemunder the given conditionswhere the major structural events take place at the millisecondtime-scale.The secondpossiblesource for damage,the powerful laser-flashhas also been shown to have no effects on the present lipids, which are relatively stable compounds,by checking for impurities in the extracted lipids with thin layer chromatographyafler the experiments.However, other lessstablelipids may well be subjectof radiation damageunder similarconditions[65]. It has been shown [2, 661 that short hydrocarbon chains, which are formed as a consequenceof radiation damagein a lamellar La-phase after extensive X-ray exposure are lowering the La - HI, transition signiIicantly, since these radiation damage products are stabilizing the H,I - phase.It is therefore highly advisableto avoid unnecessarylong exposure timesin the X-ray beamand to check the integrity of the samplenot only before but alsoafter the measurements. In this respectthe useof shorterX-ray wavelengthsshouldbe consideredasa way to reduceeffectively radiation damagebecauseof their lower absorptionby the sample[39]. Another problem, not directly relatedto radiation damagemay arisefrom the fact that the absorbedX-ray energy will eventually lead to a heating effect within the sample.At modem synchrotron sources,X-ray power densitiesin the order of 1 mWatt/mm2 can be estimated, which can lead to a heating of an aqueoussample,with the typical 1 mm thickness,by some tenths of “C/s. This may not seemdramatic, but with even higher X-ray fluxes as are to be expected with future sources,this effect will have to be taken seriously into account. Finally, however, it is justiIied to make a positive note in favour of synchrotron sources:radiation damageis primarily a dose-determinedeffect, which meansthat the sameamount of primary damageis causedby a given dose,irrespective of whether this is obtainedby low- or high-flux radiation. Thus, ifno damageoccurs during an exposureover hours at conventional sources,a correspondingexposureat a synchrotron sourcewhich takes fractions of a second,will alsobe without damage.Indeed, the shorter exposuretimesmay be even favourable, sinceall secondary reactions are diffusion controlled and therefore a quick exposuremay even lead to valid data before the secondarydestructionprocessesare complete.

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6. Conclusions The introduction of the classicalrelaxation approach using LB-temperature-jumpor hydrostatic pressure-jumptechnology to time-resolvedX-ray @action have beenproven to be suitable methods to describe dynamic structural processesat the millisecond level. The application of this method to study the mechanismsof phospholipid phase transitions has provided new insights, which cannot be gainedby near-equilibriumor slow-scanexperiments, sincethe pathways clearly difbernot only in their rates, but also qualitatively. This is related to the notion of the transition from linear to non-linear nonequilibrium phenomena and the following generalconclusionscanbe drawn: (1) The speedand efficiency of cooperative phasetransitionsis basedupon the formation of planar, lattice-equivalent interfaces which move quickly throughout the systems.Thus, individual molecular diffusion steps are minimized. This type of transition resemblesthe martensiticmechanism (2) Following this concept, symmetry-homologoustransitions are two-state martensitic processeswith transition timesin the order of millisecondsor below. (3) Dissipativeordered structnresare involved as intermediatesin symmetry-heterologous transitions. These intermediate lattices provide the connecting structural hinges between the recedingand the arisingphasestructures. (4) Thesemechanismsimply bulk deformationsof the crystal&es, which, in principle, may be relevant to locomotion in chemo- or physicotropic effects in natural systems,where multilayeredstructuresare present(e.g., light-sensitiveorganelles,skintissue,myelin).

Acknowledgements The original work cited in this article has been supportedby grants the dsterreichischer Fonds zur Fiirderung der WissenschaftlichenForschung, and the Jubilaumsfonds der GsterreichischenNationalbank. Thanksare dueto the staff of the EMBL Hamburg Outstation at DESY, in particular to G.Rapp and M.Rappolt, for their help and tiuitll

discussions.

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