slopes - revisão - coc - 2005

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CurrentOrganic Chemistry,2005,9,889-898 889

1385-2728/05 $50.00+.00 2005 Bentham Science Publishers Ltd.

Overview of Common Spectroscopic Methods to Determine theOrientation/Alignment of Membrane Probes and Drugs in Lipidic Bilayers

Slvia C. D. N. Lopes1 and Miguel A. R. B. Castanho2*

1Centro de Qumica-Fsica Molecular, Instituto Superior Tcnico, Av. Rovisco Pais, 1049-001 Lisboa, Portugal

2Centro de Qumica e Bioqumica da Faculdade de Cincias da Universidade de Lisboa, Campo Grande, Ed. C8,

1749-016 Lisboa, Portugal

Abstract: The in-depth location and orientation of membrane probes and drugs inserted in lipidic bilayers areregarded important key-properties that cannot be overlooked during molecular design and synthesis. Severalspectroscopic phenomena (e.g. excitonic interaction) and molecular recognition (e.g. ligand-receptor interaction)depend on these properties. However, molecular orientation in lipidic membranes is scarcely addressed. This paperoverviews some of the most important techniques and methodologies used to study orientation of molecules relativeto the surrounding lipidic matrix, namely: FTIR linear dichroism, UV-Vis linear dichroism, Time-resolvedfluorescence anisotropy, NMR, and Surface Plasmon Resonance.

1. INTRODUCTION

Orientation has been a relinquished aspect when studyingmembranes, receptor-mediated processes, drugs actions, andmembrane probes, over the years. Nevertheless, biologicalmembranes are dynamic structures where the orientation andthe molecular ordinance play a basic role in the maintenanceof their own functions. Moreover, molecular orientation (inbiological membranes) is a key-issue to understand immuneresponses or cellular communication, for instance(receptor/ligand interactions, in a broad sense). In medicaltechnology, for example, orientation of antibodies andenzymes determines the sensitivity and efficiency ofdiagnostic tests [1, 2].

In the membrane catalysis model, Sargent andSchwyzer proposed that peptides would interact withmembrane lipids in order to adopt the necessary

conformation for docking cell receptors [3]. This way themolecular mechanism of receptors mediated processes isbased both on receptor and on membrane requirements. Inthe case of peptides, the location and orientation of somespecific amino acid residues are crucial factors for both theinteraction with cell receptors, and biological activity (e.g.tyrosine residue in enkephalin neuropeptides [4]). Many ofthese molecular mechanisms of receptors mediated processesare related with health problems (e.g. pain and neurologicaldisorders) and one of the main research objectives is toconceive drugs that are able to eliminate, diminish and/orrelieve the symptoms. Different strategies can be used totarget drugs to its site of action: (1) natural products can bechemically modified into analogues that are able to maintain

fundamental properties but are better assimilated by theorganism, potentiating its use; (2) new chemical productscan be synthesized so they can mimic natural products and

* Address correspondence to this author at the Centro de Qumica eBioqumica da Faculdade de Cincias da Universidade de Lisboa, CampoGrande, Ed. C8, 1749-016 Lisboa, Portugal. Tel: +351 21 7500931; Fax:+351 21 7500088; E-mail: [email protected]

(3) drugs can be associated with a drug carrier system.However these drugs or analogs must mimic the natural

approach to the natural receptor with favorable orientationand correct exposure. Therefore orientation must be afundamental aspect to be considered in drug designing.

Orientational information on membrane probes is veryscarce. Most times our notion of probes orientation relativeto the membrane surface is guided by chemical intuitionbased on what is known for the bilayers structure, mainly theconformation of the acyl chain of lipids [5, 6]. Nevertheless,although sometimes chemical intuition proves realistic [7],others is illusory [8]. Depending on the membrane propertyto be reported or the phenomena involved (e.g. excitonicinteraction), probe orientation may be an important feature tobe considered during design and performance tests.

2. INFRARED SPECTROSCOPY (IR)

Although possible, transmission infrared absorptionspectroscopy is not usually used to study molecularorientation. Technical difficulties and low reproducibilityprevents its ordinary application [9]. Internal reflectiontechniques have been therefore long and more widely usedsince they circumvent most of these difficulties. Attenuatedtotal internal reflection-Fourier transform infraredspectroscopy (ATR-FTIR) methodology is one of the mostpopular and it has been used to conclude on the averagesecondary structure and orientation of biologically importantmolecules (e.g. membrane proteins) inserted in lipidmultibilayers (for reviews see references [10-12]). In atypical ATR-FTIR experiment, a trapezoidal cell (so-calledinternal reflection element (IRE), usually made ofgermanium or ZnSe), is covered with lipid multibilayerscontaining the molecule under study. These multibilayerfilms are obtained by applying an aqueous lipid suspensiononto one side of the ATR crystal and semi-drying it under agentle stream of nitrogen. The infrared beam is then focusedinto the IRE and the light crosses the plate from one side tothe other, by a series of internal reflections, originating anevanescent wave (for detailed description see references [10,


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890 Current Organic Chemistry, 2005, Vol. 9, No. 9 Lopes and Castanho

11]). This harmonic electromagnetic wave, characterized byits amplitude, and which oscillates at the same frequency asthe incident radiation, propagates along the IRE surface anddecays exponentially in the direction perpendicular to theinterface. The absorption of the energy of the resulting fieldby the lipids and other molecules there inserted (e.g.proteins) generates the ATR-FTIR spectrum which containsinformation about the sample. In macroscopically ordereddeposited membrane on a solid support, as the ones used inATR-FTIR experiments, every transition dipolar moments inthe membranes molecules have the same average orientationrelative to the normal of the IRE surface. Therefore if onechanges the orientation of the electric field component, bymeans of a polarizer, it is possible to detect orientationaldipole changes. Maximum or minimum light absorption willbe observed, if the dipole transition moment is parallel orperpendicular to the electric fields component of the incidentlight, respectively (Fig. (1)). The difference between thespectra recorded with parallel and perpendicularpolarizations is denominated dichroic spectrum and it is theone that possesses orientational information (Fig. (2)). Alarger absorbance for the parallel polarization indicates adipole preferentially oriented near the normal of the ATRplate. However if a larger absorbance is observed for theperpendicular polarization a dipole oriented parallel to theATR plate can be inferred. Nevertheless more detailedorientational information can be obtained by the calculus ofthe dichroic ratio:

R = A//A

(1)

which is the ratio between the integrated absorption of aband measured with, a parallel and a perpendicularpolarization of the incident light, respectively. This dichroicratio is related to an orientational order parameter ():

R = a + b 1+3 P2

1 P2

(2)

in which

P2 =3cos2 1

2(3)

and where a and b depend on the time average square electricfield amplitudes of the evanescent wave in the film at the

IRE/film interface and refers to the angle between thetransition dipolar moment and the normal to the cell. UsuallyATR-FTIR uses a combination of several characteristicbands to conclude about the orientation and/or secondstructure (for examples see references [12-14]). However thedipole transition moment is not always easily related to themolecular axis and sometimes is necessary to combine ATR-FTIR studies with simulation studies in order to relate them[15]. In those cases it may be convenient to convert the orderparameter obtained for the dipole transition moment to theone related to the molecular axis so that the overallmolecular orientation can be inferred. This can be achievedby using:

P2 =

3cos2 ' 1

2 P2'

(4)Where and refer to axis with relativeorientation between them. A more complex expressioncan be derived when one deals with -helix proteins, inwhich the experimental is a product of three individualorder parameters: (1) of the membrane with respect to theIRE, (2) of the helix within the membrane plane and (3) ofthe dipole orientation of amide I or amide II with respect tothe helix axis (for details see references [11, 16]).

It should be stressed that only one order parameter,, can be calculated by ATR-FTIR measurements andthat previous knowledge on the sample thickness is needed,which can be quite complex to achieve [11]. Anotherproblem is the superimposition of the vibrational bands ofthe lipidic matrix with the molecules under study that maybias or even prevent calculation. To overcome thislimitation Arkin and co-workers [17] have developed analternative method to study the structure of -helicalproteins, site-specific infrared dichroism (SSID), which canbe combined with FTIR. SSID uses oriented lipid bilayerswhich incorporated, in the original idea, -helical proteinsisotopically labelled at specific residues [17]. Samples arethen analysed using infrared polarised light in a classicalway. The method overcomes the superimposition of bands

Fig. (2). Schematic representation of the internal reflection element(IRE) used in ATR-FTIR experiments. // and denote thepolarized direction relative to the normal of the IRE surface.

Fig. (1). ATR-FTIR spectra of polyene antibiotic filipin in DLPCmembranes for parallel (A) and perpendicular (B) orientations ofthe polarizer at pH 7.4. Dichroic spectrum (C) was obtained as //minus .


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Overview of Common Spectroscopic Methods Current Organic Chemistry, 2005, Vol. 9, No. 9 891

by isotope-label shifting of a specific vibrational mode sothat its dichroism can be directly measured and which can beused in a large classes of molecules (not only proteins). Asthe spectral contribution of the chromophore depends on its

orientation, a three dimensional description of the backboneof the protein and orientation of a specific group/bond can beobtained.

Nevertheless, and regardless of some limitationspresented, ATR-FTIR has three major advantages over othertechniques, which are: (1) light scattering problems areinsignificant, (2) sometimes, depending on the molecules tobe studied, it is possible to simultaneously study thesemolecules and lipids and (3) small sample volumes (~10l)are usually needed.

3. UV-VIS LINEAR DICHROISM

In isotropic solution the molecular orientations offluorophores are randomly distributed and even if polarized

light is used what is really measured is a spatial averagesignal resulting from this distribution. However, inmacroscopically aligned membranes [18] information aboutthe orientation of the transition dipolar moment can beobtained through the dependence of linearly polarized lightabsorption on the angle formed by light polarization andsample orientation [18], which is generally called lineardichroism (LD). Sample preparation is critical in LDtechniques since disordered lipidic matrixes may introduceartifacts in the orientational distribution of biomoleculesunder study. Supported multibilayers can be obtained by

several methods as Langmuir-Blodgett (LB) technique [19,20], spin-coating [21], vesicle fusion in a quartz substrate[22], and by semi-drying of an aqueous lipid suspensionunder a gentle stream of nitrogen onto one side of the solidsupport (quartz slide in UV-Vis LD or IRE in ATR-FTIRexperiments) [11, 18]. To know details regarding sampleproperties, namely hydratation, thickness and homogeneityobtained by the different methods see references [23-26].

In a LD experiment, a quartz slide is covered with sampleand its absorption variation (A) with the measured angle(, that is the angle formed by the polarization directionrelative to the system director; Fig. (3)), using polarized lightis given by:

(5)

(sin() and the relative refractive index, n, are introduced toaccount for experimental artifacts [18]). The termsin()A/A=/2 is the dichroic ratio and is obtainedfrom equation (3) and regards the electronic absorptiontransition moment distribution relative to the normal of the

membrane. If the molecular axis and the transition momentare parallel, the molecular distribution function has the same.

LD methodology allows the calculation of another orderparameter, known as the fourth rank order parameter (). is calculated by means of a steady-state fluorescencespectroscopy having excitation and emission in a 90 anglegeometry, using polarized light [27] (Fig. (4)). If theabsorption and emission dipoles are parallel to the molecularsymmetry axis, can be calculated from:

(6)

( is the angle depicted in Figure 4; m and b depend on both and , and G, f() and n are correction factors[18, 27]). The subscripts in Iij refer to the position of theexcitation (i) or emission (j) polarizers in the lab frame (v vertical; h horizontal)). is known from absorption experiments and canbe obtained from the linear fit to the GIvh/(f().Ivv) vs. sin2data, moreover can be calculated from both the slopeand the intercept ofGIvh/(f().Ivv) vs. sin2. A specific valueof limits the range of possible (eq.7) [27].

P4 min =35 P2

2 10 P2 718

P4

5 P2 + 712

= P4 max (7)

The correction factors involved in and determination are discussed in detail in reference [27].However two significant factors should be taken in specialaccount: (1) the angular dependent propagation of lightpassing trough an interface of two isotropic media havingdifferent refraction indices, and (2) the transmission fraction

1 Reprinted from Spectroscopy Int. J., 17, M. A.R.B. Castanho, S. Lopes, M.Fernandes, Using UV-Vis. Linear dichroism to study the orientation ofmolecular probes and biomolecules in lipidic membranes, 377-398,Copyright (2003), with permission from IOS Press

Fig. (3). Schematic representation of the experimental set-up forUV-Vis. absorption LD studies in lipidic membranes (not drawn toscale). The incident beam (A), with polarization B (horizontal inthe lab frame), impinges on the quartz slide and supported bilayers(only one bilayer is represented for the sake of clarity). The anglebetween them () equals the one formed by the system director(ZZ) and the light polarization (B). variation is accomplished byrotation of the quartz slide around the axis where A and Bintersect.1

sin ()A2=

3

P2cos 2 1 +

1 n2

P2=

A

GI

h

f '()I= m sin

2 + b


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of light passing trough a boundary of two media beingdependent on its polarization state.

Since it is possible to experimentally access two orderparameters, ( and ) UV-Vis LD methodologyoffers, as major advantage, the possibility of an orientationaldistribution function (f()) calculation ( is the anglebetween the long axis of a molecule and the system director,which is the normal to the bilayer plane) and the respectiveprobability density function (f()sin()). f() can bedescribed as a Legendre polynomial series [27]:

f( ) =1

22L+ 1( ) PL PL cos( )

L e v e n

(8)

(PL(cos) are Legendre polynomials; is theensemble-average of PL (cos) and is referred to as the Lth

rank order parameter). However, and since only and can be known from experiment, the quest for

f()sin() resumes to two steps: (1) and determination (previously explained), and (2) finding anapproximated function for f()sin() from and only. The most common realistic approximation combines

the application of the maximum entropy method with theLagrange multipliers method [28] in which the resultingdistribution is the broadest possible from all the universe ofdistributions having that particular (, ) pair. (fordetails see ref [29] and Fig. (5)). Table I summarizes thedifferent types of distributions obtained for particular (,) pairs [30].

UV-Vis. LD methodologies have been applied in a largenumber of studies involving different classes of moleculessuch as membrane probes [7, 8], polyene macrolide

antibiotics [31, 32], multifunctional peptides [33, 34] andDNA-ligand systems [35].

UV-Vis. LD techniques present numerous advantagessuch as: (1) can be easily implemented, from both theinstrumental and methodological points of view, (2)instrumental adaptation, sample preparation and dataanalysis are simple, and and can be obtained forstrongly absorbing and emitting molecules, enabling a quitestraightforward estimate of the orientational densityprobability function. Moreover for samples having a smallabsorption values, obtained from other LD techniques(e.g. ATR-FTIR LD) can be used, as long as the same systemdirector is considered.

Recently fluorescence imaging of two-photon LD havebeen applied to study molecular orientation in cellmembranes [36] which represents a new insight and apromising tool in orientational studies.

4. TIME-RESOLVED FLUORESCENCE ANISO-TROPY DECAYS

Fluorescence emission anisotropy measurements can beused to obtain information on size, shape and location ofbiomolecules and/or rigidity of various molecular

environments (e.g. phospholipids) [37]. Large unilamellarvesicles (obtained by the extrusion method [38]) with

different phospholipids composition are the most commonmodel system of membranes used in these studies.Anisotropy measurements are based in a principle of

2 Reprinted from Spectroscopy Int. J., 17, M. A.R.B. Castanho, S. Lopes, M.Fernandes, Using UV-Vis. Linear dichroism to study the orientation ofmolecular probes and biomolecules in lipidic membranes, 377-398,Copyright (2003), with permission from IOS Press3 Reprinted from Biophys J., 68, M. A. Bos and J. M. Kleijn, Determinationof the orientation distribution of adsorbed fluorophores using TIRF. I.Theory, 2566-2572, Copyright (1995), with permission from ByophysicalSociety

Fig. (4). Schematic representation of the experimental set-up forfluorescence LD studies in lipidic membranes (not drawn to scale).The excitation beam (A), with polarization horizontal (H) orvertical (V; perpendicular to the figure plane) in the lab frame,

impinges on the quartz slide and supported bilayers (only one isrepresented for the sake of clarity). Fluorescence light collection(B) is carried out in a 90 angle geometry The angle between them() equals the one formed by the system director (ZZ) and the lightpolarization (B). Rotation of the quartz plate causes a variation inangle .2

Fig. (5). Interrelation between the order parameters and. The physical boundaries of and are indicated bysolid curves. The insets show the shape of the distribution function

f() for between 0 and 90 as calculated following themaximum-entropy method.3


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photoselectivity; fluorophores preferentially absorb photonswhose electric vectors are aligned parallel to its transitionmoment [37], which in turn has a defined orientation relativeto the molecular axis. In general fluorescence emissionanisotropy, r, can de defined as

r=I// I

I//+2I(9)

where I// and I are the intensities of the polarizationcomponents parallel and perpendicular to the polarization of

the excitation radiation, respectively. However theanisotropy in its functional form is:

r=Ivv GIvh

Ivv +2GIvh; G =

Ihv

Ihh(10)

(where G is a correcting factor that accounts for the differentefficiency of the detecting system for parallel andperpendicular polarized light and the two subscripts in I areused to indicate the orientation of the excitation andemission polarizers, respectively; v-vertical, h-horizontal).

Table 1. Summary of the different types of distributions obtained for different and as discussed in reference [30]

Distribution

0 0 Total disorder (random)

3cos 2c 1

2

MIN Total order fixed orientation along

c = arccos2

3P2 +

1

3

12

(conic surface distribution)

1

2cos0 1+ cos0( )( )

1

8cos0 1+ cos0( ) 7cos 20 3( )

Analogous to a particle in a conic box:

homogeneous distribution inside the cone

of surface at angle 0.

f ( ) =1

2 1+ cos0( )( )if 0 1

2P4 MAX =

5 < P2 > +7

12

When =MAX, a double function is obtained along the parallel andperpendicular orientations relative to thelipidic bilayer plan. f() is describedby

f ( ) =exp 4P4 cos( )

sen ( )exp 4P4 cos( )( )d0

(Equation 18 in reference [18] with 2=0.)


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The fluorescence anisotropy decay (anisotropy is followed ina nanosecond time scale, after pulse excitation of thefluorophores), r(t), depends both on the molecularorientational order and dynamics during the fluorophoresexcited state. At t=0, r(t), is only dependent on intrinsicspectroscopic characteristics of the fluorophore. Maximalvalue for anisotropy is r(t=0)=r0=0.4 in macroscopicallyrandom distributions of molecular orientation (i.e.macroscopically isotropic systems). r0 will decrease with

time, trough Brownian rotation, and will tend to zero in anunrestricted isotropic distribution. However, fluorophoresthat follow orientational distributions that are restricted (asmost molecules inserted in lipidic membranes) lead tolimiting anisotropies, r (r(t) in the limit t), differentfrom zero (i.e. depolarisation is not complete, Fig. (6)). r0and rare related to the second rank order parameter (r)can be obtained by the analysis of the decay trough thefollowing relation:

r = r0 P2 r2 (11)

Nevertheless this relation is only valid if: (1) thetransition moments are parallel to the symmetry axes of themolecule, and (2) if the orientational distribution function isthe same in the excited and ground state. A more generalequation would be [39]:

r = r0 P2 r P2 r* (12)

(* denotes an excited state parameter).

No information can be obtained on the orientation of thedirector axis relative to the lipidic matrix, since they areunoriented, but both equations hold true regardless of theorientation of the director axis relative to the membranesurface [18]. One of the major disadvantages of this methodis that the molecular orientation relative to the membranecannot be known; nevertheless options can be restrained (formore detailed discussion see reference [18]) and attemptshave been made to circumvent the limitation [40].

The r(t) decay from r0 to r is not only dependent onr but also dependent on the fourth rank orderparameter, r, and the rotational diffusion rate of thefluorophore around an axis perpendicular to the transitiondipole, D. Zannoni et al. [41] attained a quantitative model

for r(t), assuming that D is the same in the whole bilayer.Van der Meer et al. [42] worked out simplified approximatedequations (see equations 13 to 20), consisting of the sum ofthree exponentials plus a constant in which r, rand D can be obtained as fitting parameters in unorientedvesicles (e.g. [43]). A more detailed knowledge aboutfluorophore orientation can be obtained through anorientational distribution function which can be derived,using the same approximation used in UV-Vis LD

methodology.

r(t) = r0 gi exp( ti )i=1

3

+ g4

(13)

g1 =1

5+

2 P2 r7

+18 P4 r

35 P2 r

2 (14)

g2 =2

5+

2 P2 r7

24 P4 r

35(15)

g3 =2

5

4 P2 r

7

+6 P4 r

35

(16)

g4 = P2 r2 (17)

1 = g1 6D1

5+

P2 r7

12 P4 r

35

(18)

2 = g2 12D1

5+

P2 r14

+8 P4 r

35

(19)

3 = g3 12D1

5

P2 r7

2 P4 r

35

(20)

Unusual time-resolved fluorescence anisotropy decays,in which an initial decay is followed by a rise, are sometimesobserved (e.g. [32, 40, 44]). In general this phenomenon hasbeen interpreted as the result of microheterogeneity ofenvironments for the fluorophores, which may be a result ofa variation of local lifetime and/or local fluorescenceanisotropy.

5. RAMAN SPECTROSCOPY

Raman spectroscopy is related to changes inpolarizability associated with molecular vibrations and isusually performed with green, red or near-infrared lasers[45]. These changes give rise to scattered radiation whichcan be collected in a specific direction, usually named

analyzer direction [45]. The wavelengths, which are usuallybelow the first electronic transitions of most molecules (asassumed by the scattering theory), and intensities of thescattered light can be used to identify functional groups in amolecule. Because the intensity of the Raman signal isinversely proportional to the fourth power of the excitationwavelength, it is advantageous to use as short wavelengths aspossible. Polarized Raman spectroscopy has been used toconclude on the orientation of diverse molecules such as,single wall carbon nano tubes [46], protein [47, 48] or DNAresidues [49]. Nevertheless the most popular Raman methods

Fig. (6). Time-resolved fluorescence anisotropy decay of polyeneantibiotic filipin in aqueous solution (A) and inserted in largeunilamellar vesicles of DPPC (B), at room temperature.

B

A


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have been the ones that used the so-called resonance Ramaneffect. In fact, if the wavelength of the exciting laser iswithin the range of the electronic spectrum of a molecule, theintensity of some Raman-active vibrations increases by afactor of 102-104 (Resonance-Enhanced Raman Scattering,[50, 51]). Surface-enhanced Raman spectroscopy (SERS)has been used to study orientation of several molecules incontact with lipid monolayers deposited on planar supports[52]. Surface-enhanced Raman spectroscopy needs the

presence of a thin silver layer (15-20 nm thick) deposited onthe planar support before the transfer of the planar mono- orbilayer (through the Langmuir-Blodgett technique [19])[50-52]. These conditions makes it possible to enhance theRaman signal of deposited molecules (by six orders ofmagnitude), which otherwise could not be detected. Becauseof the short range of SERS, the observed Raman spectrum ismainly due to the monolayer in contact with silver. TheSERS effect can be increased, if the laser wavelength used isin the absorption band of the molecule under study (surface-enhanced resonance Raman scattering, SERRS). Thescattered light is then collected in a direction perpendicularto the surface of the sample and orientation can be concludefrom the analysis of characteristic bands. Moreover,

Resonance Raman spectroscopy is also a major probe of thechemistry of fullerenes, polydiacetylenes and many othermolecules which strongly absorb visible radiation [50-52]. Avariation to this technique is the deep ultra-violet resonanceRaman (UVRR) spectroscopy. UVRR uses a UV laser sourceand is a potentially powerful tool because at a wavelengthbelow 250nm fluorescence emission do not interfere theUVRR spectrum and it can provide similar informationobtained from IR without the same level of interference fromwater [53]. However one of the most powerful recentmethods to study orientation is the Raman linear intensitydifference (RLID) method. This method was first describedby Takeuchi and co-workers [54] to study orientation of anindole ring in a filamentous virus, where the authors derivedan equation relating the RLID to the orientation of theultraviolet resonance Raman (UVRR) chromophore. In RLIDthe molecules under study are aligned by hydrodynamicshear force in a flow cell composed of an outer rotatingcylinder and an inner stationary rod (flow orientation) (formore experimental details see reference [54]). The differencebetween the two resonance Raman spectral intensities,obtained by the UV laser, with parallel and perpendicularpolarizations with respect to the direction of the alignment,gives information about chromophores orientation. Thereduced RLID, , is defined as [54]:

=3 I// I( )I// +2I

=3(5cos 4 + 6cos 2 3)

2(cos4 + 3)(21)

(where is the angle of inclination of the transition momentunder study with respect to the flow orientation). Howeverthis equation only olds true if all the molecules have perfectuniaxial alignment; if not (as in most of the cases), acorrection for the fraction of randomly oriented particles asto be taken into account ([54]; equation (22)):

=15f(5cos 4 +6cos 2 3)

10f(cos4 +3)(22)

More details can be obtained in references [54-56].

One of the major advantages of Raman spectroscopy isthat it can provide similar information to the one obtained,for example from infra-red spectroscopy, without the samelevel of interference from water absorption which is ideal forstudying biological systems. However, Raman spectroscopyis not more widely used for orientation determinationbecause: (1) when visible wavelength lasers are used as theexcitation source (because they are reliable and cheap)fluorescence is also excited and this swamps the Raman

scattered signal making the measurements virtuallyimpossible, and (2) due to the high cost of lasers and opticsfor UV spectral region suitable for Raman spectroscopy.

6. SURFACE PLASMON RESONANCE

Surface plasmon resonance (SPR) has been one of themost used optical techniques for studying surface andinterfacial phenomena in pharmaceutical and analyticalchemistry research [57] structural properties (e.g. metals-electrolyte surfaces and lipid bilayers, [58]), and, morerecently, for the study of membrane biochemistry andbiophysics [59, 60]. The SPR technique exploits the fact thatlight, at certain resonance conditions, excites a wave, whichresults from collective oscillations of conducting electrons

(plasmons) in a thin metallic film, providing a source of anevanescent electromagnetic field, which can probe theoptical properties of systems in direct contact with theplasmon-generating medium [61, 62]. The resonance isachieved by varying the incident light wavelength at a fixedangle (at or above the critical angle) or by changing the angle(at a fixed wavelength) [62]. The amplitude of the plasmonelectromagnetic wave is maximal at the interface betweenthe plasmon-generating and the emergent medium (air, wateror lipid film in contact with water) [62]. In practice SPR usesa polarized laser wave to excite plasmons on a thin silver (orgold) film deposited onto the front of a glass prism. A Teflonsheet, with a central pin-hole (which is coupled to themetallic film), allows the spreading of small amounts of the

molecules/system under study, which in some controlledconditions may spontaneously orientate on the solid support.The most common solid supports are made of silver or goldand can have highly variable surfaces, includingcarboxymethylated surfaces, dextran free flat surfaces andchelated nickel surfaces (for binding His-tagged ligands).SPR makes use of the evanescent wave, which decaysexponentially with the penetration distance, to sense theoptical properties of the metal/system interface without anyinterference from the properties of the bulk volume [57, 62].The refractive index near a sensor surface, for instance, canbe used to conclude on receptor-ligand interactions andprobing mechanisms of drug/lipid membrane interactions[60]. However in the most common SPR methodology used,the plasmons are generated only by perpendicular polarizedcomponent of the excitation light relative to the film surfaceand more detailed conclusions about orientation of thesystems is prevented. With the purpose of having moredetailed information about anisotropic systems Salamon andco-workers developed a new variant of this technique, in thelate nineteens, which they called coupled plasmon-waveguide resonance (CPWR) spectroscopy [63]. CPWRcouples SPR and waveguide modes being more effective forcharacterize anisotropic biological systems (as membranes)and events which occur there.


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In CPWR the thin metal film is coated with a thickersilica layer (which is not present in SPR). As consequencethe molecules that are attached at the outer surface of the

silica, and under the appropriate experimental conditions,can be excited by light polarized perpendicular (p) or parallel(s) to the Ag-SiO2 plane. By measuring resonances with bothp and s excitation CPWR allows the study of the anisotropicoptical properties of biomembranes systems (e.g. drug-lipidor protein-lipid interactions). The degree of molecular orderis characterized by the refractive-index anisotropy (An) or bythe absorption anisotropy (Ak). An is related to the meanpolarizabilities along the p and s polarizations. Ak is relatedto the degree of order of molecular segments containing

chromophore groups that absorb at the excitation wavelength[64]. Ak is proportional to :

Ak=3

23 cos2 1( ) = 3 P2 (23)

(where is the tilt angle of the molecular principal axisrelative to the surface normal).

As in LD studies if the molecules are macroscopically

aligned, and the orientation of the transition dipoles withrespect to the molecular axes are known, the anisotropyprovides overall information about molecules orientation.

The major advantages of CPWR are: (1) the detectionmethod allows the measures to be performed in either time-resolved or steady-state modes, (2) its high sensitivity, (3)high simplicity and (4) is fast, which are crucial factorsmainly when a small quantity of the sample is available.

7. NMR

NMR has met important advancements in the domain ofmembrane-inserted peptide orientation recently [65-67].Such advancements have been thoroughly reviewed byBechinger et al. in 2004 [68], including peculiar sample

spinning experimental set-ups, which will not be covered inthis paper. Only the basics of solid-state NMR applied tostatic samples and oriented samples are within the scope ofthis review. Readers are referred to references [69] and [68]for additional information on motional averaging, rotationaldynamics and dipolar couplings, for instance, and overviewof peptides where the methodologies have been applied.Likewise, segmental orientation of flexible molecules (e.g.[70]) will not be addressed.

Considering the amide groups in peptides labeled with15N in such dilution that interactions between different labelsare negligible and that 1H and 15N nuclei are decoupled, abroad (~160 ppm) powder-pattern is observed in solid stateNMR (Fig. (7D), [68]). However under adequate orthogonal

coordinates (the principal axis system) the diagonalelements of a 33 matrix tensor (11, 22, 33; theterminology and approach of references [69] and [68] areadopted) suffice to describe the system. The tensor resultsfrom the arrangement of the nuclei and bonds in themolecular frame and can be converted into any othercoordinate systems. Namely, it is possible to relate theorientation of the tensor within the molecule with themagnetic field direction of the NMR spectrometer(laboratory macroscopic frame). The component of thechemical shift tensor projected in the magnetic fielddirection (ZZ component) is related to the experimentalNMR chemical shift value. When expressed in terms ofEuler angles and (Fig. (7E), [68]) and the elements ofthe chemical shift tensor 11, 22 and 33, the measurable zzis:zz =11 sin2 cos2 + 22 sin 2 sin2 + 33 cos2 (24)

A possible graphical depiction of the chemical shift is anellipsoid (Fig. (7E), [68]) where the length of the three mainaxes are 1/ ii (i=1, 2, 3). The intersect of the magnetic

4 Reprinted from Biochim. Biophys. Acta Biomemb., 1666, Bechinger, B.;Aisenbrey, C.; Bertani, P., The alignment, structure and dynamics ofmembrane-associated polypeptides by solid-state NMR spectroscopy,Copyright (2004), with permission from Elsevier

Fig. (7). Proton-decoupled 15N solid-state NMR spectra of the in-

plane oriented model peptide LK15 in C20-PC with alignments ofthe membrane normal parallel (A) and perpendicular (B) to themagnetic field direction. (C) Simulated powder spectra underconditions of fast rotational diffusion around the membrane normal.(D) Static simulated powder spectrum. (E) The 15N chemical shifttensor is represented as an ellipsoid. (F) Transmembrane-insertedhelical peptide is represented as a cylinder and the tilt and rotationalpitch angles and are indicated. The approximated alignment ofthe static tensor elements is shown within the helix. Fast rotationaround the bilayer normal results in averaged tensor elements //and .

4


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field vector with the ellipsoid corresponds to 1/ zz with zzbeing the apparent chemical shift at a given orientation of themolecule [68]. To convert the NMR (label) information intostructural molecular information, it is mandatory to know thepositioning of the tensor elements within the molecule. Theproton-decoupled 15N chemical shift tensors in the peptidicbond show modest variation with respect to the secondarystructure of the peptide chain ([68] and references therein).Using molecular models Bechinger and co-workers [68]have shown that the 33 tensor is oriented within a few

degrees along an -helix long axis (Fig. (7F), [68]). Figure(8) shows simulations of15N solid-state NMR obtained froma model for -helical peptide reconstituted into orientedmembranes with the membrane normal parallel to themagnetic field direction [68]. To create the simulatedspectra, Bechinger and co-workers [68] have considered allbackbone atoms of the 18-residue helices were labeled with15N. The measured 15N chemical shift is a function of boththe tilt angle () and the rotational pitch angle (, Fig. (7F),[68]).

8. FINAL REMARKS - OTHER TECHNIQUES

Other techniques have contributed to enlighten molecularorientation in lipidic matrices, although not as extensively asthe ones mentioned above. Two of them are worthremarking.

Electron spin resonance has long been used with spinlabeled molecules inserted in oriented membranes [70]. Atfirst the technique was used to gather information on thealignment of supported multibilayers and membranes butwas later used to conclude on the orientation of membraneproteins (e.g. [71]). Recent developments include themagnetical alignment of phospholipidic bicelles [72], which

have been used for instance to study the structuralorientation and dynamics of a stearic acid, in combinationwith NMR spectroscopy [70]. However, the intrinsiccomplexity of the technique associated to a scarce diversityof spin-labels hinders its generalized application.

Fluorescence resonance energy transfer (FRET)phenomena depend on the relative orientation of donor andacceptor molecules. Nevertheless, it is not usually used fororientation determination because this dependence is veryweak [73]. Moreover, orientation relative to the lipidicsurface is difficult to ascertain and the results may be biasedby the uncertainty in the donor-acceptor distance andrefractive index.

In the future, single molecule orientation will probably be

a hot topic. So far only a few experiments in very controlledconditions have succeeded. Recently fluorescence imagingof two-photon LD have been applied to study molecularorientation in cell membranes [36], which represents a newinsight and a promising tool in orientational studies.

ACKNOWLEDGEMENTS

The authors acknowledge Fundao para a Cincia eTecnologia (Portugal) for funding and grant SFRH / BD /6497 / 2001 to S. Lopes. Reproduction permission isacknowledged to IOS Press (Figures 3 and 4), BiophysicalSociety (Figure 5), Elsevier (Figures 7 and 8) and respectivearticles authors.

ABBREVIATIONS LIST

ATR-FTIR = attenuated total internal reflection-Fouriertransform infrared spectroscopy

CPWR = coupled plasmon-waveguide resonanceDLPC = dilauroylphosphatidylcholineDPPC = dipalmitoylphosphatidylcholine

5 Reprinted from Biochim. Biophys. Acta Biomemb., 1666, Bechinger, B.;Aisenbrey, C.; Bertani, P., The alignment, structure and dynamics ofmembrane-associated polypeptides by solid-state NMR spectroscopy,Copyright (2004), with permission from Elsevier

Fig. (8). Simulations of proton-decoupled 15N chemical shiftspectra of helices oriented with their long axes at the anglesindicated. The sum of the spectra from the individual amide sites isshown a single site label will, therefore, be found within theanisotropic chemical shift dispersion indicated. During thesimulations, the static main tensor elements were 223, 75 and 61ppm with an alignment of the tensor elements within the molecularframe.5


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FRET = fluorescence resonance energy transferIRE = internal reflection elementLD = linear dichroismLUV = large unilamellar vesicleNMR = nuclear magnetic resonanceRLID = Raman linear intensity differenceSERRS = surface-enhanced resonance Raman

scattering

SERS = surface-enhanced resonance RamanspectroscopySPR = surface plasmon resonanceSSID = site-specific infrared dichroismUVRR = ultra-violet resonance RamanUV-Vis = utra-violet-visible

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