Proc Indian Natn Sci Acad, 69, A, No. 1, January 2003, pp. 25-35© Printed in India.

NOVEL INSIGHTS INTO MEMBRANE PROTEIN STRUCTUREAND DYNAMICS UTILIZING THE WAVELENGTH-SELECTIVE

FLUORESCENCE APPROACH

H RAGHURAMAN, DEVAKI A KELKAR AND AMITABHA CHATTOPADHYAY*Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad-500 007 (India)

(Received 06 November 2002; Accepted 08 November 2002)

Wavelength-selective fluorescence comprises a set of approaches based on the red edge effect in -fluorescencespectroscopy which can be used to directly monitor the environment and dynamics around a fluorophore in acomplex biological system. A shift in the wavelength of maximum fluorescence emission toward higher wavelengths,caused by a shift in the excitation wavelength toward the red edge of absorption band, is termed red edge excitationshift (REES). This effect is mostly observed with polar fluorophores in motionally restricted media such as veryviscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophoreis comparable to or longer than its fluorescence lifetime. REES arises from slow rates of solvent relaxation (reorientation)around an excited state fluorophore which is a function of the motional restriction imposed on the solvent moleculesin the immediate vicinity of the fluorophore. Utilizing this approach, it becomes possible to probe the mobilityparameters of the environment itself (which is represented by the relaxing solvent molecules) using the fluorophoremerely as a reporter group. Further, since the ubiquitous solvent for biological systems is water, the informationobtained in such cases will come from the otherwise ‘optically silent’ water molecules. This makes REES andrelated techniques extremely useful since hydration plays a crucial modulatory role in a large number of importantcellular events including lipid-protein interactions and ion transport. The application of the wavelength-selectivefluorescence approach as a powerful tool to monitor organization and dynamics of membrane proteins and peptidesis discussed in this review.

Key Words: Wavelength-Selective Fluorescence Approach; Red Edge Excitation Shift (REES);Membrane Proteins and Peptides; Tryptophan Fluorescence; Hydration Dynamics; SolventDipole Relaxation

1 Introduction

Biological membranes are complex assemblies oflipids and proteins that allow cellularcompartmentalization and act as the interfacethrough which cells communicate with each otherand with the external milieu. The biologicalmembrane constitutes the site of many importantcellular functions. However, our understanding ofthese processes at the molecular level is limited bythe lack of high resolution three dimensionalstructures of membrane-bound molecules. It isextremely difficult to crystallize membrane-bound proteins or peptides for diffraction studies.Although the first complete X-ray crystallographicanalysis of an integral membrane protein wassuccessfully carried out a number of yearsback1, the number of membrane proteins whose

* e-mail: amit@ccmb.res.in Fax: +91-40-2716-0311

X-ray crystal structures are known is still verysmall and represent only ~ 0.2% of all solvedprotein structures2. This is in spite of the fact thatabout 30-40% of all proteins are integralmembrane proteins3. The great disparity betweenour understanding of soluble proteins andmembrane proteins is a consequence of manypractical problems of working with membraneproteins. Even high resolution NMR methodshave limited applications for membrane-boundproteins and peptides because of slow reorientationtimes in membranes4.

Due to the inherent difficulty in crystallizingmembrane proteins, most structural analyses ofsuch molecules have utilized other biophysicaltechniques with an emphasis on spectroscopicapproaches. Spectroscopic techniques whichprovide both structural and dynamic informationtherefore become very useful for analyses of

H RAGHURAMAN et al.

membrane proteins. Fluorescence spectroscopyrepresents one such approach and is widely usedin analysis of membrane protein structure andfunction. The advantages of using fluorescencetechniques are intrinsic sensitivity, suitable timescale, non-invasive nature, and minimumperturbation 5-8. This review is focussed on theapplication of a novel approach, the wavelength-selective fluorescence approach, as a powerful toolto monitor the organization and dynamics of,membrane proteins and peptides.

Wavelength-selective fluorescence comprises aset of approaches based on the red edge effect-influorescence spectroscopy which can be used todirectly monitor the environment and dynamicsaround a fluorophore in a complex biologicalsystem. A shift in the wavelength of maximumfluorescence emission toward higher wavelengths,caused by a shift in the excitation wavelengthtoward the red edge of absorption band, is termedred edge excitation shift (REES)8-10. This effect ismostly observed with polar fluorophores inmotionally restricted media such as very viscoussolutions or condensed phases where the dipolarrelaxation time for the solvent shell around afluorophore is comparable to or longer than itsfluorescence lifetime. REES arises from slow ratesof solvent relaxation (reorientation) around anexcited state fluorophore which is a function of themotional restriction imposed on the solvent moleculesin the immediate vicinity of the fluorophore. Thisapproach therefore allows one to probe the mobilityparameters of the environment itself (dynamics ofsolvation) represented by the relaxing solventmolecules using the fluorophore merely as areporter group. Further, since the ubiquitous solventfor biological systems is water, the informationobtained in such cases will come from the otherwise‘optically silent’, water molecules. This makes REESand related techniques extremely useful sincehydration plays a crucial modulatory role in a largenumber of important cellular events. The applicationof the wavelength-selective fluorescence approachto membranes and membrane-mimetic systems hasbeen recently reviewed8,9.

2 Red Edge Excitation Shift (REES)

In general, fluorescence emission is governed byKasha’s rule which states that fluorescence normallyoccurs from the zero vibrational level of the first

excited electronic state of a mo1ecule11,12. It isobvious from this rule that fluorescence should beindependent / of the wavelength of excitation. Infact, such a lack of dependence of fluorescenceemission parameters on excitation wavelength isoften taken as a criterion for purity and homogeneityof a molecule. Thus, for a fluorophore in a bulknon-viscous solvent, the fluorescence decay ratesand the wavelength of maximum emission areusually independent of the excitation wavelength.

However, this generalization breaks down incase of polar fluorophores in motionally restrictedmedia such as very viscous solutions or condensedphases, that is, when the mobility of the surroundingmatrix relative to the fluorophore is considerablyreduced. This situation arises because of theimportance of the solvent shell and its dynamicsaround the fluorophore during the process ofabsorption of a photon and its subsequent emissionas fluorescence (see later). Under such conditions,when the excitation wavelength is gradually shiftedto the red edge- of the absorption band, themaximum of fluorescence emission exhibits aconcomitant shift toward higher wavelengths. Sucha shift in the wavelength of maximum emissiontoward higher wavelengths, caused by acorresponding shift in the excitation wavelengthtoward the red edge of the absorption band, istermed the Red Edge Excitation Shift (REES)8,13,15.Since REES is observed only under conditions ofrestricted mobility, it serves as a reliable indicatorof the dynamics of fluorophore environment.

The genesis of REES lies in the change influorophore-solvent interactions in the ground andexcited states brought about by a change in thedipole moment of the fluorophore upon excitation,and the rate at which solvent molecules reorientaround the excited state fluorophore14,16,22. For apolar fluorophore, there exists a statisticaldistribution of solvation states based on theirdipolar interactions with the solvent molecules bothin the ground and excited states in order to minimizethe energy of the given state. Since the dipolemoment of a molecule changes upon excitation23, ,the solvent dipoles have to reorient around this newexcited state dipole moment of the fluorophore, soas to attain an energetically favourable orientation.This readjustment of the dipolar interaction of thesolvent molecules with the fluorophore essentiallyconsists of two components. First, the redistribution

26

NOVEL INSIGHTS INTO MEMBRANE PROTEIN STRUCTURE AND DYNAMICS 27

of electrons in the surrounding solvent moleculesbecause of the altered dipole moment of the excitedstate fluorophore, and then the physical reorientationof the solvent molecules around the excited statefluorophore. The former process is almostinstantaneous, i.e., electron redistribution in solventmolecules occurs at about the same time scale asthe process of excitation of the fluorophore itself(10-15 sec). The reorientation of the solvent dipoles,however, requires a net physical displacement. Itis thus a much slower process and is dependent onthe restriction to their mobility as offered by thesurrounding matrix. More precisely, for a polarfluorophore in a bulk non-viscous solvent, thisreorientation time τ

S is of the order of 10-12 sec,so that all the solvent molecules completely reorientaround the excited state dipole of the fluorophorewell within its excited state lifetime τ

F which is

typically of the order of 10-9 seca. Hence, irrespectiveof the excitation wavelength used, all emission isobserved only from the solvent-relaxed state.However, if the same fluorophore is now placedin a viscous medium, this reorientation process isslowed down such that the solvent reorientationtime (τ

S) is of the order of 10-9 sec or longer. Under

these conditions, excitation by progressively lowerenergy quanta, i.e., excitation wavelength beinggradually shifted toward the red edge of theabsorption band, selectively excites thosefluorophores which interact more strongly with thesolvent molecules in the excited state. These arethe fluorophores around which the solvent moleculesare oriented in such a way as to be more similarto that found in the solvent-relaxed state. Thus, thenecessary condition for giving rise to REES is thata different average population of fluorophore isexcited at each excitation wavelength and,more importantly, that the difference is maintainedin the, time scale of fluorescence lifetime. Asdiscussed above, this requires that the dipolarrelaxation time for the solvent shall be comparableto or longer than the fluorescence lifetime so thatfluorescence occurs from various partiallyrelaxed states. This implies a reduced mobility ofthe surrounding matrix with respect to thefluorophore.

The essential criteria for the observation of thered edge effect can therefore be summarized asfollows: (i) the fluorophore should normally bepolar so as to be able to suitably orient theneighbouring solvent molecules in the ground state;(ii) the solvent molecules surrounding thefluorophore should be polar; (iii) the solventreorientation time (τ

S) around the excited state

dipole moment of the fluorophore should becomparable to or longer than the fluorescencelifetime (τ

F) and (iv) there should be a relatively

large change in the dipole moment of the fluorophoreupon excitation. The observed spectral shifts thusdepend both on the properties of the fluorophoreitself (i.e., the vectorial difference between thedipole moments in the ground and excited states),and also on properties of the environment interactingwith it (which is a function of the solvent reorientationtime τ

S). It has previously been shown by us that

for 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD)-labelled phospholipids incorporated into modelmembranes, a dipole moment change of ~ 4 D uponexcitation is enough to give rise to significant rededge effects23 A recent comprehensive review on.the red edge effect is provided by Demchenko24.

3 The Two State Model

The two state model provides a simple conceptualframework within which one can consider thephenomenon of solvent relaxation18~25-27. This modelassumes that fluorescence emission proceeds fromtwo discontinuous or discrete states (see Fig. 1):the initial or Franck-Condon (F) state and the finalor relaxed (R) state. Excitation at the maximum(center) of the absorption band (λ

C), initially yields

the Franck-Condon excited state around whichsolvent reorientation has not taken place. Thisinitially excited state F can then decay to acompletely relaxed state R where solventreorientation around the excited state fluorophoreis complete, with a characteristic relaxation time τ

S.

The rate of this relaxation is determined by bothgeneral and specific interactions between thefluorophore and the surrounding solvent moleculesand the rate at which these interactions are modifiedin response to the newly created excited state dipole

a Use of a single parameter τS to describe the relaxation of solvent molecules is a first order approximation since a set of relaxation

times would exist in real systems.7 However, such an approximation is often made to make the relaxation model simple. Thus, τS

may be considered as a simple effective parameter characterizing the solvent relaxation process.

28 H RAGHURAMAN et al.

Condition Excitation at λc Excitatlon at λR

λ1 λ2 λ1 λ2

Emission Wavelength

Fig. 1 A schematic representation of the two state model of solventrelaxation in terms of energy levels (top) and fluorescenceemission spectra (bottom). F and R states refer to theFranck-Condon (or initial) and relaxed (or final) excitedstates. τS is the reorientation time of the solvent moleculesand τF is the lifetime of the fluorophore. The large arrowsrepresent the dipole moment vectors of the fluorophore andthe small arrows denote those of the solvent moleculessurrounding the fluorophore. λC and λR are the respectivewavelengths associated withdirectexcitation of the Franck-Condon and relaxed states. See text (Section 3) for otherdetails.

moment. If we denote the direct excitation of theF state as λC and such excitation of the R state asλR (where λR > λC), then there could be threepossibilities (Fig. 1). If the rate of solvent reorien-tation is much faster than the fluorescence lifetime

(τF >>τS), the solvent molecules have enough timeto reorient before the fluorophore emits. Therefore,irrespective of the excitation wavelength used (λC

or λR), the relaxed emission is observed. In otherwords, under the conditions described above,excitation of either the F state or the R state yieldsemission spectra centered a t λ2. Thissituation is observed for a fluorophore in bulk, non-viscous solvents or at high temperatures. On theother hand, when the fluorophore is placed in aviscous medium or condensed phase or is frozen

to very low temperatures, the relaxation time, whichis a function of the rate of the physical reorientationof the solvent molecules, is drastically increasedand is much longer than the fluorescence lifetime(τF << τS ). Under these conditions, the blue shiftedemission of the F state (emission maximum at λ1) is observed with central excitation (λC) However,if the same system is now excited with lower energyquanta (λR) it will select a subclass of the totalfluorophore population around which the solventdipoles are oriented so as to decrease the energydifference between the ground and the excitedstates. In particular, this photoselected ground stateis higher in energy and the photoselected excitedstate is at a lower energy level (see Fig. 1) becauseof the alignment of the solvent dipoles in the‘solvent-relaxed’ orientation. Therefore, red edgeexcitation under these conditions selects thosemolecules which have a solvent-relaxed environmentand resultantly, red shifted absorption and emissionspectra (i.e., the emission spectrum is centered atλ2 under these conditions). The third possibility isthe situation where the rate of solvent reorientationis comparable to the fluorescence lifetime i.e., under conditions of intermediate viscosity ortemperature. In this case, excitation at λC gives riseto a spectrum centered at a wavelength intermediatebetween λ1 and λ2. This can be attributed to thefact that although the F state alone is initiallyexcited, the comparable values of τF and τS giverise to emissions from both F and R states. Thisinhomogeneous spectrum thus also has a widerspectral distribution27-29. Upon red edge excitation,the emission occurs mainly from the R state, givingrise to a more red shifted emission spectrum whichis also comparatively narrow, as it is composed ofemission predominantly from the R state17,19.

4 The Continuous Model

This model was first proposed by Bakhshiev andco-workers30-32 to provide a phenomenologicaldescription of time-dependent effects ofsolvent-fluorophore interactions on emission spectrain terms of the observed spectral parameters (seeFig. 2). According to this model, the maximum offluorescence emission [or more rigorously, thecenter of gravity of the emission spectrum, vm (t)]is assumed to shift to lower energy in an exponentialmanner following excitation, with a characteristicrelaxation time τS, i.e.,

(τF ≈ τS)

NOVEL INSIGHTS INTO MEMBRANE PROTEIN STRUCTURE AND DYNAMICS 29

F State-

Fig. 2 The continuous model of solvent relaxation. The I staterefers to one of the intermediate states between the initial(F) and the final (R) states, in which the solvent moleculesare partially relaxed. νo, νI and V represent the frequenciescorresponding to the initially excited (Franck-Condon),intermediate, and completely relaxed states, respectively,while λC, λI, and λR denote the wavelength maximaassociated with these states. τS is the reorientation time ofthe solvent molecules. See text (Section 4) for other details.

νm (t) = ν + (νo - ν) exp (- t /τS) ... (1)where νo and ν represent the emission maxima (incm-1) of the initially excited (Franck-Condon) andthe completely relaxed states. The spectral shapeof the emission is assumed to remain constantduring the time course of the emission. Due to theexponential nature of eq. (1), the emission maximashift continuously from ν = νo at t = 0 to ν = νat t = . Therefore at t = τS In 2, the spectral shiftis 50% complete.

However, in reality, there exists a statisticaldistribution of solvation states for an ensemble ofpolar fluorophores in solution, based on theirdipolar interactions with the solvent molecules inboth the ground and excited states. The moleculesinteracting in solution may differ in their mutualorientation and interaction energies which resultsin alteration of the energies of electronic transitions.Both the character of the energy distribution at themoment of excitation and its change with time(relaxation) will determine the spectroscopicbehaviour of the system. Such a steady statespectrum will therefore be quite broad due tocontributions from the various partially relaxedsubstates. This has been referred to as the ‘dipole-

orientational broadening’ of the spectrum17,20,22,33.Taking all these into account, i.e., the statisticaldistribution of interaction energies for moleculeswith their environments, and photoselection ofexcited species according to the energy of theabsorbed or emitted quanta, Demchenko laterproposed a more realistic mode117,20,22.

5 The Wavelength-Selective FluorescenceApproach

In addition to the dependence of fluorescenceemission maxima on the excitation wavelength(REES), fluorescence polarization and lifetime arealso known to depend on the excitation andemission wavelengths in viscous solutions and inotherwise motionally restricted media. Takentogether, these constitute the wavelength-selectivefluorescence approach which consists of a set ofapproaches based on the red edge effect influorescence spectroscopy which can be used todirectly monitor the environment and dynamicsaround a fluorophore in a complex biologicalsystem8-10. Early applications of REES andwavelength-selective fluorescence to systems ofbiological relevance have been restricted mainly toindole, tryptophan, and other fluorescent probes inviscous solvents21.

6 The Unique Role of Tryptophan Residues inMembrane Proteins and Peptides: Tryptophan

and the Membrane Interface

The presence of tryptophan residues as intrinsicfluorophores in most peptides and proteins makesthem an obv ious cho i ce fo r f l uo re scencespectroscopic analyses of such systems. The roleof tryptophan residues in the structure and functionof membrane proteins has recently attracted a lotof attention34,35. The biological membrane providesa unique environment to membrane-spanningproteins and peptides thus influencing their structureand function. Membrane-spanning proteins havedistinct stretches of hydrophobic amino acids thatform the membrane-spanning domain and havebeen reported to have a significantly highertryptophan content than soluble proteins36. Inaddition, it is becoming increasingly evident thattryptophan residues in integral membraneproteins and peptides are not uniformly distributedand that they tend to be localized toward themembrane interface, possibly because they are

∞ ∞

involved in hydrogen bonding37 with the lipidcarbonyl groups or interfacial water molecules(see Fig. 3). For instance, crystal structures ofmembrane proteins such as the potassium channel38

bacteriorhodopsin39, maltoporin40 and others haveshown that most tryptophans are located in a sad dle -like ‘aromatic belt’ around the membrane interfacialregion. Statistical studies of sequence databases andavailable crystal structures of integral membraneproteins also show preferential clustering oftryptophan residues at the membrane interface34 ,41 ,42.Furthermore, for synthetic transmembrane peptides,tryptophan has been found to be an efficient anchorat the membrane intterface 4 3 and defines thehydrophobic length of transmembrane helices44.Interestingly, the role of tryptophan residues inmaintaining the structure and function of membraneprot e ins34 has attracted considerable attention. Theimportance of tryptophan residues in maintaining

the fun c tion of m emb rane proteins is exemplifiedby the fact that substitution or deletion of tryptophansoften results in reduction or loss of proteinfunctionality45,46.

30 H RAGHURAMAN et a l.

The preferential location of tryptophan residuesat the membrane interface is thought to be due tothe aromaticity of the indole moiety and the overallamphipathic nature of tryptophan47. The tryptophanrich aromatic belt at the membrane interface intransmembrane helices are thought to stabilize thehelix with respect to the membrane environment41.The tryptophan residue has a large indole side chainthat consists of two fused aromatic rings. Inmolecular terms, tryptophan is a unique amino acidsince it is capable of both hydrophobic and polarinteractions. In fact, the hydrophobicity oftryptophan. measured by partitioning into bulksolvents, has previously been shown to be dependenton the scale chosen48. Tryptophan ranks as one ofthe most hydrophobic amino acids on the basis ofits partitioning into polar solvents such as octanol49

while scales based on partitioning into nonpolarsolvents like cyclohexane50 rank it as onlyintermediate in hydrophobicity. This ambiguityresults from the fact that while tryptophan has thepolar -NH group which is capable of forminghydrogen bonds, it also has the largest nonpolar

Fig. 3 A schematic rcpresentation of the mcmbranc bilayer showing the various regions of the bilaycr displaying motional anisotropy.The membrane lipids shown have two hydrophobic tails with a phosphatidylcholine (PC) headgroup. The preferred locationsof various amino acids present in a membrane-spanning transmembrane domain are also shown. It is worth noting that the fluorescenttryptophan residues are localized in the membrane interface region, a region characterizcd by unique organization, dynamics,hydration and functionality. See text (Section 6) for other details. Adapted and modified from rcfs. [10] and [34].

NOVEL INSIGHTS INTO MEMBRANE PROTEIN STRUCTURE AND DYNAMICS 31

accessible surface area among the naturally occuringamino acid51. Wimley and White have recentlyshown from partitioning of model peptides tomembrane interfaces that the experimentallydetermined interfacial hydrophobicity of tryptophanis highest among the naturally occurring amino acidresidues thus accounting for its specific interfaciallocalization in membrane-bound peptides andproteins52. Due to its aromaticity, the tryptophanresidue is capable of π-π interactions and of weaklypolar interactions53. The amphipathic character oftryptophan gives rise to its hydrogen bondingability which could account for its orientation inmembrane proteins and its function through long-range electrostatic interaction46. The amphipathiccharacter of tryptophan also explains its interfaciallocalization in membranes due to its tendency tobe solubilized in this region of the membrane,besides favourable electrostatic interactions andhydrogen bonding.

A direct consequence of highly organizedmolecular assemblies such as membranes is therestriction imposed on the mobility of moleculesincorporated in such assemblies. It is well knownthat interiors of biological membranes are viscous,with the effective viscosity comparable to that oflight oil54,55. The biological membrane, with itsviscous interior, and distinct motional gradientalong its vertical axis, thus provides an ideal systemfor the utilization of REES in particular andwavelength-selective fluorescence in general tostudy organization and dynamics of membrane-bound proteins and peptides. The use of thisapproach becomes all the more relevant in viewof the fact that no detailed crystallographic databasefor membrane-bound proteins and peptides existsto date due to the inherent difficulty in crystallizingsuch molecules. Moreover, any information obtainedhas a dynamic component generally absent in X-ray crystallographic data.

The membrane exhibits a considerable degreeof anisotropy along the axis perpendicular to themembrane plane. Properties such as polarity, fluidity,segmental motion, ability to form hydrogen bondsand extent of water penetration vary in a depth-dependent manner in the membrane. The interfacialregion in membranes is therefore characterized byunique motional and dielectric characteristics distinctfrom both the bulk aqueous phase and the hydro-carbon-like interior of the membrane (Fig. 3)56-63.

It is a chemically heterogeneous region composedof lipid headgroups, water and portions of the acylchain64. Overall, the interfacial region of themembrane accounts for 50% of the thermal thicknessof the bilayer60. This specific region of the membraneexhibits slow rates of solvent relaxation and is alsoknown to participate in intermolecular chargeinteractions65 and hydrogen bonding through thepolar headgroup66-68. These structural features whichslow down the rate of solvent reorientation havepreviously been recognized as typical features ofsolvents giving rise to significant red edgeeffectsi6. It is therefore the membrane interfacewhich is most likely to display red edge effects andis sensitive to wavelength-selective fluorescencemeasurements69. This makes membrane peptidesand proteins ideally suited for studies using thewavelength-selective fluorescence approach sinceas discussed above, the fluorescent tryptophanresidues are localized in this region of the membrane.

7 Application of the Wavelength-SelectiveFluorescence Approach to Membrane Peptides

The membrane environment and the relative positionwithin the membrane have a profound influenceon the dynamics of amino acid residues of membranespanning helices. Both natural and syntheticmembrane peptides offer a convenient tool to studythe specific role of membrane on the orientation,incorporation, stability and function of suchpeptides70-73.

Tryptophan octyl ester (TOE) has beenrecognized as an important model for membrane-bound tryptophan residues35,74-76. The fluorescencecharacteristics of TOE incorporated into modelmembranes and membrane-mimetic systems havebeen shown to be similar to that of membrane-bound tryptophans. Consistent with the preferredinterfacial location of the tryptophan residue andthe solvent restricted environment of the interface,TOE has been found to exhibit REES. Further, theextent of REES was found to be dependent on pHindicating more motional freedom on deprotonationat higher pH35.

Melittin, the major toxic component in thevenom of the European honey bee, Apis mellifera,was one of the earlier membrane. peptides studiedusing the wavelength-selective fluorescenceapproach. Melittin is an amphipathic cationichemolytic peptide with a single functionally

32 H RAGHURAMAN et al.

important tryptophan residue”. Amphipathicity ischaracteristic of many membrane-boundpeptides and putative transmembrane helices ofmembrane proteins77,78. This has made melittin verypopular as a simple model to study lipid-proteininteractions in membranes77,79. Results fromwavelength-selective fluorescence studiesshowed that when bound to zwitterionic membranes,the micro-environment of the sole functionallyactive tryptophan of melittin was motionallyrestricted as evident from REES and other resultsin agreement with the interfacial location of thetryptophan residue80. Interestingly, wavelength-selective fluorescence studies indicate that themicroenvironment of the tryptophan residue inmelittin is modulated when bound to negativelycharged membranes, and this could be related tothe functional difference in the lytic activity of thepeptide observed in the two cases81. REES istherefore sensitive to changes in dynamics ofhydration caused by varying electrostaticinteractions. In addition, REES of melittin boundto membranes (or membrane-mimetic systems)was found to be dependent on the lipid compositionof the membrane and the hydration state ofmelittin82,83.

In yet another study, the phenomenon of REES,-in conjunction. with time-resolved fluorescencespectroscopic parameters such as wavelength-dependent fluorescence lifetimes and time-resolvedemission spectra (TRES) was utilized to study thelocalization and dynamics of the functionallyimportant tryptophan residues in the gramicidinchannel84. Gramicidin belongs to a family ofprototypical channel formers which are naturallyfluorescent due to the presence of four tryptophanresidues85. These interfacially localized tryptopbansare known to play a crucial role in the organizationand function of the channe145,46. The results fromthe above study point out the motional restrictionexperienced by the tryptophans at the peptide-lipid interface of the gramicidin channel. This isconsistent with other studies45,46 in which suchrestrictions are thought to be imposed due tohydrogen bonding between the indole rings of thetryptophan residues in the channel conformationand the neighboring lipid carbonyls. The significanceof such organization in terms of functioning of thechannel is brought out by the fact that substitution,photodamage, or chemical modification of these

tryptophans are known to give rise to channelswith altered conformation and reducedconductivity45,46,86-88. More importantly, REES andrelated fluorescence approaches could be used todistinguish between the channel and non-channelconformation of gramicidin in membranes89,90.REES of gramicidin is therefore sensitive to theconformation adopted by the peptide in themembrane. This provides a convenient spectro-scopic handle to monitor functional status of thisimportant ion channel peptide. Further, REES ofgramicidin incorporated into membranes was foundto be dependent on the lipid composition91 andphase state of the membrane92.

Wavelength-selective fluorescence approachhas also been used to monitor the membraneinteraction of synthetic peptides corresponding tothe functionally important regions of membranebinding proteins93,94. The 579-601 fragment of theectodomain of the HIV- 1 gp41 protein which isessential for its activity, was shown to incorporatein model membranes93. Upon red edge excitation,there was a substantial red shift in fluorescenceindicating a motionally restricted environment forthe single tryptophan of this peptide. The REESresult is somewhat unusual not only due to the ratherlarge magnitude of REES (18 nm) but also due tothe observation of REES for a peptide with theemission maximum at 348 nm when excited at theabsorption maximum. This is in contrast with thepreviously accepted notion that REES can only befound for Trp residue emitting between 325 and341 nm95. Large magnitude of REES (28 nm) hasalso been observed for the reporter tryptophan inmembrane-bound PT-( 1-46)F4W synthetic peptidewhich corresponds to the o-loop region of thehuman prothrombin γ-carboxyglutamic aciddomain94. In both these cases, the peptides werefound to partition to the inter-facial region of themembrane. The observed REES, would thereforebe a direct consequence of the motionally restrictedmembrane interface%. A large REES (25 nm) is alsodisplayed by the NBD-labelled peptide fragmentof apolipoprotein C-II (apoC-II19-39) when boundto egg yolk phosphatidylcholine membranes97.This observation, along with results from time-resolved fluorescence measurements, points outthat the NBD-labelled peptide is located near thepolar-inter-facial region of the bilayer where itexperiences a heterogeneous environment.

NOVEL INSIGHTS INTO MEMBRANE PROTEIN STRUCTURE AND DYNAMICS 33

The interaction of the peptide corresponding tothe γM4 transmembrane domain of the nicotinicacetylcholine receptor with membranes has recentlybeen investigated98. Wavelength-selectivefluorescence showed that the helix is oriented insuch a way that the N-terminal tryptophan (Trp 453)is located in a motionally restricted environment atthe membrane interface, confirming the previousobservations that γM4 forms a transmembranehelix99. The topology and bilayer location oftryptophan residues in the colicin El channelpeptide has very recently been explored using arather novel approach in which REES of singletryptophan containing channel peptides wasdetermined in the soluble aqueous state and whenbound to membranes100. From the difference inREES values of the channel peptides in themembrane-bound and soluble state, the topologyof the peptide was mapped out.

8 Application of the Wavelength-SelectiveFluorescence Approach to Membrane Proteins

An early application of the wavelength-selectivefluorescence approach to study membrane proteinorganization consisted of studying themicroconformational heterogeneity of the membranebinding domain of cytochrome b5 by comparingthe information obtained from the native proteinand its mutant which has a single tryptophan residuein this domain101. Both these proteins show a redshift in the emission spectrum when excited at thelong wavelength edge of the excitation spectrumindicating thereby that the tryptophan residue(s) inboth cases are localized in a region of motionalconstraint.

In a later study, the tryptophans of the pore-forming Staphylococcus aureus α-toxin wereshown to exhibit REES102. However, nosignificant difference was observed between REESdisplayed by the soluble and the membrane-boundforms of the toxin indicating no drastic change inthe tryptophan environment upon membranebinding. The tryptophans in the E. coli porin Omp-F, a pore forming channel protein, also exhibitsREES 103.

Membrane active proteins that exist in solubleand membrane-bound forms undergo considerableconformational change in the transition from thesoluble to the membrane-bound state. One suchprotein is the mitochondrial creatine kinase which

shows a large increase in REES upon membranebinding104. The soluble form exhibits REES of 6nm which increases to a REES of 19 nm in thepresence of membranes which is indicative of arestricted environment for the tryptophans in themembrane-bound form. Since the tryptophanresidues are localized in the protein interior, theobserved motional restriction has been attributedto conformational change of the protein inducedby lipid binding.

9 Conclusions and Future Perspectives

Water plays a crucial role in the formation andmaintenance of cellular architecture. Knowledge ofdynamics of hydration at the molecular level is thusof considerable importance in understanding cellularstructure and function105-112. As mentioned earlier,REES is based on the change in fluorophore-solventinteractions in the ground and excited states broughtabout by a change in the dipole moment of thefluorophore upon excitation, and the rate at whichsolvent molecules reorient around the excited statefluorophore. Since for biological systems, theubiquitous solvent is water, the information obtainedin such cases will come from the otherwise ‘opticallysilent’ water molecules. The unique feature aboutREES is that while all other fluorescence techniquessuch as fluorescence quenching, energy transferand polarization measurements yield informationabout the fluorophore (either intrinsic or extrinsic)itself, REES provides information about the relativerates of solvent (water in biological systems)relaxation dynamics which is not possible to obtainby other techniques. This makes the use of REESand the wavelength-selective fluorescence approachpowerful in membrane biology since hydrationplays a crucial modulatory role in a large numberof cellular events involving the membrane such aslipid-protein interactions107 and ion transport105-107.REES and the wavelength-selective fluorescenceapproach, in combination with the novel molecularbiology approaches for site-specific incorporationof unnatural fluorophores at specific sites113, couldprove to be an extremely powerful tool to probeorganization and dynamics of membrane proteinsand peptides.

10 Acknowledgements

Work in author’s (AC) laboratory was supported bythe Council of Scientific and Industrial Research

34 H RAGHURAMAN et al.

and Department of Science and Technology, of the work described in this review article wasGovernment of India. The authors (HR and DAK) carried out by former and present members of Dr.are thankful to the University Grants Commission Chattopadhyay’s group whose contributions arefor the award of Senior Research Fellowships. Some gratefully acknowledged.

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