Organization and Dynamics of Membrane Probes ... - ACS Publications

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Organization and Dynamics of Membrane Probes and Proteins Utilizing the Red Edge Excitation Shift Sourav Haldar, Arunima Chaudhuri, and Amitabha Chattopadhyay* Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007 India ABSTRACT: Dynamics of confined water has interesting implications in the organization and function of molecular assemblies such as membranes. A direct consequence of this type of organization is the restriction imposed on the mobility of the constituent structural units. Interestingly, this restriction (confinement) of mobility couples the motion of solvent (water) molecules with the slow moving molecules in the assembly. It is in this context that the red edge excitation shift (REES) represents a sensitive approach to monitor the environment and dynamics around a fluorophore in such organized assemblies. 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 the absorption band, is termed REES. REES relies on slow solvent reorientation in the excited state of a fluorophore that can be used to monitor the environment and dynamics around a fluorophore in a host assembly. In this article, we focus on the application of REES to monitor organization and dynamics of membrane probes and proteins.

1. INTRODUCTION Molecular confinement brings about interesting properties in water in terms of its organization and dynamics which are very different from that experienced in bulk water.1,2 Although water is the most abundant liquid on the surface of the earth, it is often found in a captive environment.3 There are numerous examples in geochemistry, tribology, nanofluidics, and biology (such as the interfacial region in membranes, crevices on protein surface, and grooves of DNA) where water is often found in confinement. In fact, cellular water in biology has been referred to as tamed hydra.4 Confined water molecules present in biomolecules and interfaces are sometimes described as biological water.2,5 Interfacial confinement destroys the water
water hydrogen bond network and couples the motion of water molecules with the slow moving macromolecules.2 Since most biologically important molecular events take place at the biomolecule
solvent interface,6,7 the dynamics of solvent (water) in interfacial confinement has interesting consequences on the function of biomolecules.5,8 Solvent relaxation measurements provide a spectroscopic window to monitor the local dynamics (flexibility) of solvent (water) molecules at biological interfaces.2 Several spectroscopic approaches have been developed to explore solvent relaxation dynamics. These include time-dependent fluorescence stokes shift (TDFSS),2,5,8 three-photon echo peak shift (3PEPS),9 nuclear magnetic relaxation dispersion (NMRD),6 and twodimensional infrared spectroscopy.10 Recently, computer simulation approaches have been used for solvent relaxation studies.11
14 Organized molecular assemblies (such as membranes) can be considered as large cooperative units with characteristics very different from the individual structural units which constitute r 2011 American Chemical Society

them. A direct consequence of such organization is the restriction imposed on the mobility of their constituent structural units. Interestingly, restriction (confinement) helps to couple the motion of solvent (water) molecules with the slow moving molecules in the host assembly.2 In this context, red edge excitation shift (REES) represents a unique approach which relies on slow solvent reorientation in the excited state of a fluorophore that can be used to monitor the environment and dynamics around a fluorophore in an organized molecular assembly.15
24 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 the absorption band, is termed red edge excitation shift (REES). REES arises from relatively slow rates (compared to fluorescence lifetime) of solvent relaxation (reorientation) around an excited state fluorophore. REES therefore depends on the environment-induced motional restriction imposed on the solvent molecules in the immediate vicinity of the fluorophore. This approach allows us to assess the rotational mobility of the environment itself (which is represented by the relaxing solvent molecules) utilizing the fluorophore merely as a reporter group (the definition of solvent in this context is rather pragmatic; solvent relaxation dynamics includes dynamics of restricted solvent (water) as well as the dynamics of the host dipolar matrix such as the peptide backbone in proteins).25 This review focuses on the application of REES to monitor the organization and dynamics of membrane probes and proteins.

Received: January 10, 2011 Revised: February 13, 2011 Published: March 23, 2011 5693

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2. REES: CONCEPTUAL FRAMEWORK In general, fluorescence emission is governed by Kasha’s rule which states that fluorescence normally occurs from the lowest vibrational level of the first excited electronic state of a molecule.26,27 It is obvious from this rule that fluorescence emission, in principle, should be independent of wavelength of excitation. In fact, such a lack of dependence of fluorescence emission on excitation wavelength is often taken as a criterion for purity and homogeneity of a molecule. In the case of a fluorophore in a bulk nonviscous solvent, the fluorescence decay rates and the wavelength of maximum emission are therefore usually independent of the excitation wavelength. This generalization breaks down in the case of polar fluorophores in a motionally restricted environment such as viscous solutions or condensed phases (e.g., polymer films, gels, glass, ionic liquids, proteins, and membranes), that is, when the mobility of the surrounding matrix relative to the fluorophore is considerably reduced. This situation arises because of the importance of the solvent shell and its dynamics around the fluorophore during the process of absorption of a photon and its subsequent emission as fluorescence (see below). Under such spatiotemporally constrained conditions, when the excitation wavelength is gradually shifted to the red edge of the absorption band, the fluorescence emission maximum exhibits a concomitant shift toward higher wavelengths. As mentioned earlier, such a shift in the wavelength of the emission maximum toward higher wavelengths, induced by a corresponding shift in the excitation wavelength toward the red edge of the absorption band, is termed REES.15
24 Since REES is observed only under conditions of restricted mobility, it serves as a reliable indicator of the local dynamics of the fluorophore environment. The molecular origin of REES lies in altered fluorophore
solvent interactions in the ground and excited states caused by a change in the dipole moment of the fluorophore upon excitation and the rate at which solvent molecules reorient around the excited state fluorophore. For a polar fluorophore, a statistical distribution of solvation states exists based on their dipolar interactions with the solvent molecules in both the ground and excited states. Since the dipole moment of a molecule changes upon excitation, the solvent dipoles need to reorient around this new excited state dipole of the fluorophore, to attain an energetically favorable orientation. This readjustment of the dipolar interaction of the solvent molecules with the fluorophore consists of two components: (i) the redistribution of electrons in the surrounding solvent molecules (electronic polarization of the environment) because of the altered dipole moment of the excited state fluorophore, (ii) followed by the physical reorientation of the solvent molecules around the excited state fluorophore. The former process is almost instantaneous; i.e., electron redistribution in solvent molecules occurs at about the same time scale as the process of excitation of the fluorophore itself (10
15 s). On the other hand, the reorientation of solvent dipoles requires a net physical displacement and is therefore a much slower process. The latter process is dependent on the restriction to their mobility offered by the surrounding matrix. More precisely, for a polar fluorophore in a bulk nonviscous solvent, this reorientation occurs at a time scale of the order of 10
12 s, so that all solvent molecules completely reorient around the excited state dipole of the fluorophore well within its excited state lifetime, which is typically of the order of 10
9 s. In such a case, all emission is therefore observed only from the solvent-relaxed state, irrespective of the excitation wavelength

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used. However, if the same fluorophore is placed in a spatiotemporally constrained viscous medium, this reorientation process is slowed down to 10
9 s or longer. Under these conditions, excitation by progressively lower energy quanta, i.e., the excitation wavelength being gradually shifted toward the red edge of the absorption band, selectively excites the subpopulation of fluorophores that interact more strongly with the solvent molecules in the excited state. These are the fluorophores around which the solvent molecules are oriented in a manner similar to that found in the solvent-relaxed state. The necessary condition for giving rise to REES is therefore that a different average population is excited at each excitation wavelength and, more importantly, that the difference is maintained in the time scale of fluorescence lifetime. As discussed above, this requires that the dipolar relaxation time for the solvent shell be comparable to or longer than the fluorescence lifetime, so that fluorescence occurs from various partially relaxed states. This implies a reduced mobility of the surrounding matrix with respect to the fluorophore. If the solvent relaxation dynamics is fast compared to the rate of emission, the rapid interconversion between the substates with different orientation of dipoles will result in the loss of dependence of emission maximum on excitation wavelength. REES is generally observed with polar fluorophores in motionally restricted media such as very viscous solutions or condensed phases where the dipolar relaxation time for the solvent shell around a fluorophore is comparable to or longer than its fluorescence lifetime. The essential criteria for the observation of the red edge effect can therefore be summarized as follows: (a) the fluorophore should normally be polar to be able to suitably orient the neighboring solvent molecules in the ground state (however, molecules such as bianthryl which are nonpolar in the ground state and yet could be polar in the excited state due to intramolecular charge transfer reaction are exceptions to this28,29); (b) the “solvent” molecules (such as water, protein backbone, lipid headgroups) surrounding the fluorophore should be polar; (c) the solvent reorientation time around the excited state dipole should be comparable to or longer than the fluorescence lifetime (this is the most important criterion of REES); and (d) there should be a relatively large change in the dipole moment of the fluorophore upon excitation. In general, the dipole moment of molecules increases upon excitation.30 Although less common, dipole moment has been shown to decrease in the excited state for some probes. Nonetheless, these probes are capable of exhibiting REES (e.g., the dipole moment of a merocyanine dye decreases by ∼5.5 D upon excitation).29,31 Taken together, the observed spectral shifts therefore depend both on the properties of the fluorophore itself (i.e., the difference between the dipole moments in the ground and excited states) and on dynamic properties of the environment (medium) interacting with it. (a). Two-State Model. The two -state model provides a simple conceptual framework within which solvent relaxation can be considered.15,22,32
34 This model assumes that fluorescence emission takes place from two discontinuous or discrete states (see Figure 1): the initial or Franck
Condon (FC) state and the final or solvent-relaxed (R) state. Excitation at the maximum (center) of the absorption band (λC) initially gives rise to the Franck
Condon excited state (the so-called “vertically excited state”) around which solvent reorientation has not taken place. This initially excited state (FC) can then decay to a completely solvent relaxed state (R) where solvent reorientation around the excited state fluorophore is complete, with a characteristic relaxation time (τs).35 The rate of solvent relaxation is determined by 5694

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Figure 1. Schematic representation of the two-state model of solvent relaxation in terms of energy levels (top) and spectra (bottom). FC and R states refer to the Franck
Condon (or initial) excited and solvent relaxed (or final) states, respectively. The reorientation time of solvent molecules and the lifetime of the fluorophore are denoted as τS and τF, respectively. The thick arrows represent the dipole moment vectors of the fluorophore, and the small ellipses around them denote solvent molecules surrounding the fluorophore. Directionality of the elliptical shapes denotes the orientation of solvent molecules. The orientation of solvent molecules in the FC state is identical to that in the ground state, while they are different in the solvent relaxed states. The respective wavelengths associated with direct excitation of the Franck
Condon and solvent relaxed states are denoted as λC and λR, respectively. Red edge excitation photoselects a subpopulation of fluorophores in the ground state around which the solvent molecules are oriented much like the solvent relaxed excited state of the fluorophore. See text for other details. Adapted and modified from ref 15.

both general and specific interactions between the fluorophore and surrounding solvent molecules and the rate at which these interactions are modified in response to the newly created excited state dipole. If λC corresponds to the wavelength of excitation for direct excitation of the FC state and λR is the corresponding excitation wavelength for excitation of the R state (where λR > λC), then there could be three possibilities (see Figure 1). If the rate of solvent reorientation is much faster than fluorescence lifetime (i.e., τS , τF), the solvent molecules would have enough time to

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reorient before the fluorophore emits. In such a case, emission from the solvent-relaxed state would be observed, irrespective of the excitation wavelength used (λC or λR). In other words, under this condition, excitation of either the FC state or the R state yields emission spectra centered at λ2 (see Figure 1). This criterion is fulfilled in the case of a fluorophore in bulk, nonviscous solvents or at high temperatures. On the other hand, when the fluorophore is placed in a viscous medium or condensed phase or is frozen to very low temperatures (under spatiotemporally constrained condition), the relaxation time τS (which is a function of the rate of the physical reorientation of the solvent molecules) is drastically increased and is now much longer than the fluorescence lifetime, i.e., τS . τF. Under this condition, the blue-shifted emission of the FC state (emission maximum at λ1) is observed with central excitation (λC). However, upon excitation with lower-energy quanta (λR), a subset of the total fluorophore population would be selected, around which the solvent dipoles are oriented to decrease the energy difference between the ground and the excited states. This means that upon excitation with longer wavelength (λR) the photoselected ground state is higher in energy and the photoselected excited state is at a lower energy level (see Figure 1) because of the alignment of solvent dipoles in the “solvent-relaxed” orientation. Red edge excitation under these conditions therefore selects a subpopulation of molecules which have a solvent-relaxed environment and, resultantly, red-shifted absorption and emission spectra (i.e., the emission spectrum in such a condition is centered at λ2). The third possibility arises when the rate of solvent reorientation is comparable to fluorescence lifetime (τF ≈ τS), i.e., under conditions of intermediate viscosity or temperature. In this scenario, excitation at λC gives rise to a spectrum centered at a wavelength intermediate between λ1 and λ2. This can be attributed to the fact that although the FC state alone is initially excited the comparable values of τF and τS would allow emissions from both FC and R states. Interestingly, this inhomogeneous spectrum has a wider spectral distribution.22,36,37 Upon red edge excitation, emission occurs mainly from the R state, giving rise to a more redshifted emission spectrum, which is also comparatively narrow, as it is composed of emission predominantly from the R state.38,39 (b). Continuous Model. The continuous model was first proposed by Bakhshiev and co-workers40
42 to provide a phenomenological description of time-dependent effects of solvent
fluorophore interactions on emission spectra in terms of the observed spectral parameters (see Figure 2). According to this model, the maximum of fluorescence emission [or strictly speaking, the center of gravity of the emission spectrum ν hm(t)] is assumed to shift to lower energy in an exponential fashion following excitation, with a characteristic relaxation time τS, i.e. νm ðtÞ ¼ ν¥ þ ðν0
ν¥ Þexpð
t=τS Þ

ð1Þ

where νh0 and νh¥ represent the emission maxima (in cm
1) of the initially excited (Franck
Condon) and the completely solventrelaxed states, respectively. The spectral shape of the emission is assumed to remain constant during the time course of the emission. Due to the exponential nature of eq 1, the emission maxima shift continuously from νh = νh0 at t = 0 to νh = ν h¥ at t = ¥. A time-dependent correlation function C(t) can then be introduced such that C(t) = [νhm(t)
νh¥]/[νh0
νh¥] which is a convenient measure of spectral shift. Therefore, at t = τS ln 2, the spectral shift C(t) is 50% complete. However, in real situations, based on their dipolar interactions with the solvent molecules in both the ground and excited states, 5695

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Figure 2. Continuous model of solvent relaxation. The I state refers to one of the intermediate states between the initial (Franck
Condon) and the final (solvent relaxed) states, in which solvent molecules are partially relaxed. The frequencies of transitions from ground states to the Franck
Condon, intermediate, and solvent-relaxed states are denoted as ν0, νI, and ν¥, respectively. The corresponding wavelength maxima are denoted as λC, λI, and λR. The reorientation time of the solvent is τS. See text for other details. Adapted and modified from ref 15.

a statistical distribution of solvation states for an ensemble of polar fluorophores in solution would exist. The molecules interacting in solution may differ in their mutual orientation and interaction energies, which results in alteration of the energies of electronic transitions. Both the character of the energy distribution at the moment of excitation and its change with time (relaxation) will determine the spectroscopic behavior of the system. Such a steady state spectrum will therefore be quite broad due to contributions from the various partially relaxed substates. This has been referred to as “dipole-orientational broadening” of the spectrum. Taking all these into account, i.e., the statistical distribution of interaction energies for molecules with their environments, and photoselection of excited species according to the energy of the absorbed or emitted quanta, Demchenko later proposed a more realistic model.23 In addition to REES, fluorophores localized in spatiotemporally constrained situations display wavelength-dependent anisotropy and lifetime.15

3. BIOLOGICAL MEMBRANE: AN APPROPRIATE MOLECULAR ASSEMBLY FOR REES Biological membranes are complex, two-dimensional, noncovalent, anisotropic assemblies of lipids and proteins that allow cellular compartmentalization and act as the interface through which cells communicate with each other and with the external milieu. The physical principle underlying the formation of membranes is entropic, i.e., the hydrophobic effect.43,44 Membranes impart an identity to the cell and represent an ideal milieu for the proper function of membrane proteins and constitute the site of many important cellular functions. However, our understanding of these processes at the molecular level is limited by the lack of highresolution three-dimensional structures of membrane-bound molecules and the challenges involved in obtaining dynamic information over a large range of spatiotemporal scales, characteristic of the motion of lipids and proteins in the membrane.45 As mentioned above, an obvious consequence of a high degree of organization in assemblies such as membranes is the restriction imposed on the mobility of the constituent structural units. It is well-known that interiors of biological membranes offer

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restriction to motion (i.e., viscous), with the “effective viscosity” comparable to that of light oil.46,47 The biological membrane, with its viscous interior and distinct motional gradient along its vertical axis (see below), therefore provides an appropriate molecular assembly for the application of REES to explore membrane phenomena. This becomes all the more relevant in the overall context of the difficulty encountered in crystallizing membrane-bound molecules in their native conditions.48 The interfacial region in membranes is characterized by unique motional and dielectric characteristics49 different from the bulk aqueous phase and the more isotropic hydrocarbon-like deeper regions of the membrane. The membrane interface plays an important role in functional aspects such as substrate recognition and activity of lipolytic enzymes.50 The confined water molecules at the membrane interfacial region become ordered due to the reduced possibility of energetically favorable hydrogen bonding arising out of geometrical constraints.51 This specific region of the membrane exhibits slow rates of solvent relaxation and is also known to participate in intermolecular charge interactions52 and hydrogen bonding through the polar lipid headgroup.53,54 These structural features that slow down the rate of solvent reorientation have previously been recognized as typical features of solvents giving rise to significant REES effect.55 It is therefore the membrane interface that is most likely to display the REES effect (see Figures 3A and 4A). Interestingly, the choice of a suitably localized probe is crucial in REES measurements in membranes. It is essential that the probe is polar and able to strongly partition into the membrane and intercalate with membrane lipids (see Figure 3A). In addition, the fluorescent moiety should be suitably embedded in the membrane. It is also preferable that the membrane-embedded molecule has only one fluorescent group characterized by a unique location in the membrane bilayer (not a distribution of locations). An advantage of using such fine-tuned probes is that such probes can be conveniently used to correlate the extent of REES with specific fluorophore environments in the membrane (see Section 3a below and Figure 4).56 A probe that satisfies these conditions well is the widely used lipid probe N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine (NBD-PE).57 The 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group is an extensively used fluorophore in biophysical, biochemical, and cell biological studies. NBD-labeled lipids are extensively used as fluorescent analogues of native lipids in biological and model membranes to study a variety of processes.58,59 The NBD moiety possesses some of the most desirable properties for serving as an excellent probe for both spectroscopic and microscopic applications. In NBD-PE, the fluorescent NBD label is covalently attached to the headgroup of a phosphatidylethanolamine molecule. The precise orientation and location of the NBD group of this molecule in the membrane has previously been worked out.60
65 This group has been found to be localized at the membrane interface characterized by unique motional and dielectric properties, distinct from both the bulk aqueous phase and the hydrocarbon-like interior of the membrane, therefore making it an ideal probe for monitoring red edge effects.18 In addition, previous electrophoretic measurements have shown that the NBD group in NBD-PE is uncharged at neutral pH in the membrane.61 This ensures that the NBD group does not project into the external aqueous phase. This is advantageous since the ability of a fluorophore to exhibit red edge effects is dependent on its precise location in the membrane (see below). The change in dipole moment of the NBD group upon excitation, a necessary condition for a fluorophore to exhibit 5696

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Figure 3. (A) Schematic representation of a leaflet of the membrane bilayer showing the localization of the NBD group of NBD-PE in the phosphatidylcholine bilayer. The NBD group of NBD-PE localizes at the membrane interfacial region (see text). Note that this is a time-averaged location of the NBD group (in nanosecond time scale), and fluctuations at shorter time scales are possible. The horizontal line at the bottom indicates the center of the bilayer. Adapted and modified from ref 24. Panels B
E show wavelength-selective fluorescence of NBD-PE in DOPC membranes (adapted and modified from ref 57). (B) Representative intensity-normalized fluorescence emission spectra of NBD-PE at different excitation wavelengths. The excitation wavelengths used were 465 (—), 500 (

), and 510 (- - -) nm. (C) The effect of changing excitation wavelength on the wavelength of maximum emission (REES) of NBD-PE. (D) Steady-state fluorescence anisotropy of NBD-PE with increasing excitation wavelength. (E) Intensity-averaged lifetime of NBD-PE with increasing excitation wavelength. See refs 15 and 57 for further details.

REES, has been found to be ∼4 D.30 The change in emission maximum with changing excitation wavelength (REES) and related wavelength-selective fluorescence properties of NBD-PE in dioleoyl-sn-glycero-3-phosphocholine (DOPC) membranes are shown in Figure 3 (panels B
E).57,65,66 Since the precise localization of the fluorescent NBD group in membrane-bound NBD-PE is known to be interfacial,60
65 these results imply that the interfacial region of the membrane offers considerable restriction to the reorientational motion of the solvent dipoles around the excited state fluorophore. We later showed that REES of NBD-PE can be conveniently used to monitor environmental restriction in the interface region of related assemblies such as micelles and reverse micelles.67
69 We discuss below applications of REES in exploring the organization and dynamics of lipids (probes) and proteins in biomembranes.

(a). Depth-Dependent Solvent Relaxation. Biological membranes exhibit a considerable degree of anisotropy along the axis perpendicular to the membrane plane (see Figure 4A).18,70 While the center of the bilayer is nearly isotropic, the upper portion, only a few angstroms away toward the membrane surface, is highly ordered.18,56 As a result, properties such as polarity, fluidity, segmental motion, ability to form hydrogen bonds, and extent of solvent penetration vary in a depth-dependent manner in the membrane. A direct consequence of such an anisotropic transmembrane environment is the differential extents to which the mobility of solvent (water) molecules will be retarded at different depths in the membrane compared to their mobility in the bulk aqueous phase.56 This feature gives rise to the possibility of using REES as a unique approach to explore the dynamics of differentially localized reporter fluorophores along the membrane z-axis 5697

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Figure 4. (A) Schematic representation of half of the membrane bilayer showing the anisotropy in polarity and dynamics of the bilayer. The dotted horizontal line at the bottom indicates the center of the bilayer. The membrane anisotropy along the axis perpendicular to the plane of the bilayer (z-axis) divides the membrane leaflet into three broad regions exhibiting very different dynamics (as reported by spectroscopic techniques such as ESR, NMR, and fluorescence). Region A: bulk aqueous phase characterized by fast solvent relaxation. Region B: interface, characterized by slow (restricted) solvent relaxation, hydrogen bonding (important for functionality), water penetration (interfacial water), highly heterogeneous nature. Region C: bulk hydrocarbon-like environment, isotropic, fast solvent relaxation. A polarity (dielectric) gradient is also set up along this axis (see ref 70). Fluorescent probes and peptides localized in the interfacial region B are most likely to display REES. The localizations of the anthroyloxy groups of 2- and 12-AS in phosphatidylcholine bilayers are also shown. (B) Depth-dependent REES of anthroyloxy probes: effect of changing excitation wavelength on the wavelength of maximum emission for 2-AS (9) and 12-AS (2). The anthroyloxy group is localized at the interfacial region in the case of 2-AS, while it resides in the bulk hydrocarbon region in the case of 12-AS. Adapted and modified from ref 56.

(i.e., as a membrane dipstick). We tested this idea by demonstrating that chemically identical fluorophores, varying solely in terms of their localization at different depths in the membrane, experience very different local environments, as judged by REES and related parameters.56,66 Anthroyloxy stearic acid derivatives in which the anthroyloxy group had previously been found to be either shallow 2-(9-anthroyloxy)stearic acid (2-AS) or deep 12-(9anthroyloxy)stearic acid (12-AS) were used for this purpose (see Figure 4). Anthroyloxy fatty acids are particularly well suited for this type of study since they have been found to be located at a graded series of depths in the bilayer, depending on the position of the attachment of the anthroyloxy group to the fatty acyl chain.71,72 It has been previously shown that the depth of the anthroyloxy group is almost linearly related to the number of carbon atoms between it and the carboxyl group.64 Depth analysis using the parallax method has earlier shown that the anthroyloxy probes in 2-AS (the shallow probe) and 12-AS (the deep probe) are localized at 15.8 and 6 Å from the center of the bilayer, respectively (see Figure 4A).64 Interestingly, we showed that the anthroyloxy moiety of 2- and 12-AS experiences different local membrane

microenvironments, as reflected by depth-dependent variation of REES and related wavelength-dependent fluorescence parameters. While the shallow anthroyloxy group in 2-AS exhibits a REES of 5 nm, the deeper anthroyloxy group in 12-AS did not exhibit any REES.56 These results were attributed to differential rates of solvent reorientation in the immediate vicinity of the anthroyloxy group as a function of its membrane penetration depth (i.e., slower solvent relaxation at the membrane interface relative to deeper regions). REES therefore constitutes a unique approach to monitor dynamics at defined depths in the membrane and can be conveniently used as a dipstick to characterize the depth of penetration of membrane-embedded fluorophores. (b). Interfacial Chemistry. Membrane interface chemistry can vary depending on the type of linkage (e.g., ester or ether) of the hydrocarbon chains to the glycerol backbone region. This could influence the organization and dynamics of the membrane interface.73 For example, the conformational and structural differences between ester- and ether-linked phospholipids are due to the replacement of the ester sp2 carbon of the carbonyl group with an sp3 carbon of the ether linkage.74 The additional water molecules 5698

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Figure 6. REES depends on the phase state of the membrane. The magnitudes of REES of membrane-bound DChol in gel, liquid-ordered, and fluid (liquid-disordered) phase membranes are shown. The structure of DChol is also shown. See text for other details. Adapted and modified from ref 87.

Figure 5. (A) Chemical structures of ester- (DPPC) and ether-linked (DHPC) phospholipids and the fluorescent membrane probe 2-AS. The ester and ether linkages in DPPC and DHPC (the only difference between the two phospholipids) are highlighted. (B) REES depends on interfacial chemistry: effect of changing excitation wavelength on the wavelength of maximum emission of 2-AS in DPPC (9) and DHPC (b) membranes. Adapted and modified from ref 73.

from increased hydration in ether phospholipids75
78 may replace the space occupied by the carbonyl groups in ester phospholipids.77 In addition, ether phospholipids lack carbonyl groups at the site of attachment to the glycerol backbone. As a consequence, there is reduced hydrogen bonding between ether phospholipids which allows tighter packing of the hydrocarbon chains.79 We explored the change in organization and dynamics of a membrane probe (2-AS) when the interfacial chemistry of the phospholipid constituting the host membrane is changed from ester- to ether-type linkage (with no other change in the host lipid structure).73 For this purpose, we used 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dihexadecyl-sn-glycero-3-phosphocholine (DHPC) (see Figure 5A) since they differ only in their interfacial chemistry (i.e., ester vs ether linkage). The magnitude of REES obtained for the interfacial probe (2-AS) appears to vary with the chemical nature of the linkage in the phospholipid (see Figure 5B). In other words, the rate of solvent relaxation in ester-linked membranes was found to be slow compared to the rate of solvent reorientation in ether-linked membranes. This difference was attributed to the change in the microenvironment experienced by the probe in these two cases induced by the change in interfacial chemistry of the two phospholipids. This differential dynamics

observed in ester- and ether-linked lipids assumes relevance since ether-linked phospholipids are functionally important lipids found in halophilic bacteria, cardiac tissue, and the central nervous system80 and in light of the functional significance of ether lipids in animal models.81 Interestingly, the magnitude of REES was also found to be dependent on the charge of the membrane.82 (c). Membrane Phase. The membrane phase represents an important determinant, particularly in terms of domain formation.83 In addition, hydration (water penetration) depends on the phase state of the membrane.84 Apart from gel (solid) and fluid (liquid-disordered) phases, membranes (in the presence of cholesterol) are found to exist in a separate phase, namely, the liquid-ordered phase.85 While the lipid acyl chains are ordered and extended in all-trans conformation in the gel phase, they are fluid and disordered in the liquid-disordered phase. The liquidordered phase exists above a threshold level of cholesterol for binary lipid mixtures. In the case of DPPC membranes containing ∼40 mol % cholesterol, it is evident from the temperature
composition phase diagram that the only phase that exists is the liquid-ordered phase (i.e., no coexisting phases).83,86 This phase is characterized by acyl chains which are extended and ordered (like the gel phase) but exhibit high lateral mobility similar to the liquid-disordered phase. To explore solvent relaxation dynamics in membranes of varying phase, we monitored REES of a novel fluorescent analogue of cholesterol, 6-dansylcholestanol (DChol; see Figure 6).87 The orientation and localization of DChol in membranes are consistent with the localization of cholesterol. Interestingly, the magnitude of REES exhibited by DChol was found to be dependent on the phase state of the membrane.87 REES of DChol was found to be maximum in the fluid phase, followed by REES in the liquid-ordered and gel phases (see Figure 6). REES exhibited by DChol was found to be minimum in the gel phase. Importantly, although REES has been earlier reported in liquid-disordered and gel phase membranes,66,88 these results constituted the first report of REES in liquid-ordered membranes. This is important since membrane domains (such as “rafts”) are believed to exist in the liquid-ordered phase.85

4. REES OF MEMBRANE PROTEINS AND PEPTIDES Membrane proteins mediate a wide range of essential cellular processes such as signaling across the membrane, cell
cell 5699

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The Journal of Physical Chemistry B recognition, and membrane transport. About 30% of all open reading frames (ORFs) are predicted to encode membrane proteins, and almost 50% of all proteins encoded by eukaryotic genomes are membrane proteins.89,90 Importantly, they represent prime candidates for the generation of novel drugs in all clinical areas91,92 owing to their involvement in a wide variety of cellular processes. Membrane proteins are difficult to crystallize in their native conditions due to the intrinsic dependence on surrounding membrane lipids.48 Since a significant portion of any integral membrane protein remains in contact with the membrane lipid environment,93 membrane lipids are necessary to maintain their structure and function. It is in this context that approaches based on fluorescence spectroscopy have proved useful in elucidating the organization, topology, and orientation of membrane proteins and peptides.94,95 Most structural studies of membrane proteins have primarily focused on transmembrane R-helices in terms of their amino acid composition and packing interactions. Unfortunately, the parts of the proteins (or peptides) located at the membrane interface region have received much less attention.96 Application of REES to membrane proteins and peptides using their native tryptophan fluorescence has helped address this lacuna (see below). The biological membrane provides a unique environment to membrane-spanning proteins and peptides thereby influencing their structure and function. Membrane-spanning proteins have distinct stretches of hydrophobic amino acids that form the membrane-spanning domain and are reported to have a significantly higher tryptophan content than soluble proteins.97 The presence of tryptophan residues as intrinsic fluorophores in membrane peptides and proteins makes them an obvious choice for fluorescence spectroscopic analyses. The role of tryptophan residues in the structure and function of membrane proteins and peptides has attracted a lot of attention.20,98
102 It appears that tryptophan residues in integral membrane proteins and peptides are not uniformly distributed, and they tend to be localized toward the membrane interface, possibly because they are involved in hydrogen bonding103 with the lipid carbonyl groups or interfacial water molecules. As mentioned above (Section 3), the interfacial region in membranes is characterized by unique motional and dielectric characteristics distinct from both the bulk aqueous phase and the hydrocarbon-like interior of the membrane. The tryptophan residue has a large indole side chain that consists of two fused aromatic rings. In molecular terms, tryptophan is a unique amino acid since it is capable of both hydrophobic and polar interactions. This is due to the fact that while tryptophan has the polar
NH group which is capable of forming hydrogen bonds it also has the largest nonpolar accessible surface area among the naturally occurring amino acids.104 From partitioning of model peptides to membrane interfaces, it has been shown that the experimentally determined interfacial hydrophobicity of tryptophan is highest among the naturally occurring amino acid residues, thereby accounting for its specific interfacial localization in membrane peptides and proteins.105 In addition, the tryptophan residue is capable of π
π interactions and of weakly polar interactions due to its aromaticity.106 The amphipathic character of tryptophan gives rise to its hydrogen bonding ability which could account for its orientation in membrane proteins and its function through long-range electrostatic interaction.107,108 The amphipathic character of tryptophan also explains its interfacial localization in membranes due to its tendency to be solubilized in this region of the membrane, in addition to favorable electrostatic interactions and hydrogen

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bonding. It has already been mentioned that the membrane interface is most likely to display REES (see Section 3) and is sensitive to wavelength-selective fluorescence measurements. For this reason, the study of membrane peptides and proteins utilizing REES is particularly advantageous. Importantly, the role of tryptophan residues in maintaining the structure and function of membrane proteins is exemplified by the fact that substitution or deletion of tryptophans often results in reduction or loss of protein functionality.107,109
111 We have utilized REES to monitor the conformation and dynamics of a variety of membrane peptides and proteins such as melittin,112,113 gramicidin,114,115 the pore-forming R-toxin from S. aureus,116 and the N-terminal domain of CXC chemokine receptor (CXCR1).117 We have chosen to discuss below the application of REES to representative membrane peptides such as melittin and gramicidin. (a). Melittin. Melittin is the principal toxic component in the venom of the European honey bee Apis mellifera and is a cationic, hemolytic peptide. It is a small linear peptide composed of 26 amino acid residues in which the amino-terminal region is predominantly hydrophobic, whereas the carboxy-terminal region is hydrophilic due to the presence of a stretch of positively charged amino acids. This amphiphilic property of melittin makes it watersoluble, and yet it spontaneously associates with natural and artificial membranes and membrane-mimetics.118 These features contribute to make melittin an ideal peptide to explore the organization and dynamics of amphiphilic peripheral membrane proteins. Melittin adopts predominantly random coil conformation as a monomer in aqueous solution.119 Interestingly, melittin adopts an R-helical conformation when bound to membranes.120 Melittin is intrinsically fluorescent due to the presence of a single tryptophan residue, Trp-19, which makes it a sensitive probe to study the interaction of melittin with membranes and membrane
mimetic systems.112,121 Results from our laboratory have earlier shown that when bound to zwitterionic membranes the microenvironment of the sole functionally important tryptophan of melittin is motionally restricted, as evident from REES and other wavelength-selective fluorescence results.112,122 However, when bound to negatively charged membranes, studies employing REES indicated that the microenvironment of the tryptophan gets modulated, and this could be related to the functional difference in the lytic activity of the peptide observed in the two cases.112 Interestingly, the extent of REES of membrane-bound melittin was found to be dependent on the lipid composition (unsaturation) and ionic strength.123,124 A limitation of utilizing tryptophan fluorescence is that the information essentially originates from the carboxy terminal region of melittin, and information about the amino terminal region is lacking. The lack of information about the amino terminal region of melittin makes it difficult to comment about the orientation of melittin in membranes. To address this issue, we generated two analogues of melittin where the N-terminal glycine is covalently labeled with fluorescent tags such as NBD125 and dansyl120 groups. The results obtained using these analogues demonstrated that both NBD and dansyl groups at the amino terminal experience motional restriction and exhibit REES. Interestingly, the dansyl group at the amino terminal also exhibited dynamic Stokes’ shift, a hallmark of slow solvent relaxation.120 (b). Gramicidin. The linear peptide gramicidin forms prototypical ion channels specific for monovalent cations and has been used extensively to study the organization, dynamics, and function of membrane-spanning channels. Gramicidin is a multitryptophan protein (Trp-9, 11, 13, and 15) which serves as an excellent 5700

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Figure 7. (A) Schematic representation of the channel and nonchannel conformations of gramicidin indicating the location of tryptophan residues in the membrane bilayer. The tryptophan residues are clustered toward the membrane interface in the channel conformation, whereas they are distributed along the membrane axis in the nonchannel conformation. The membrane axis is represented by a double-headed arrow. See text for further details. (B) Conformation of membrane-bound gramicidin is sensitive to REES: effect of changing excitation wavelength on the wavelength of maximum emission for the nonchannel (`, olive), intermediate I (2, maroon), intermediate II (9, cyan), and channel (b, blue) conformations of gramicidin. Adapted and modified from ref 115.

model for transmembrane channels due to its small size, ready availability, and the relative ease with which chemical modifications can be performed.126,127 This makes gramicidin unique among small membrane-active peptides and provides the basis for its use to explore the principles that govern the folding and function of membrane-spanning channels in particular and membrane proteins in general. The unique sequence of alternating L- and D-chirality renders gramicidin sensitive to the environment in which it is placed. Gramicidin therefore adopts a wide range of environment-dependent conformations. Gramicidin has been shown to assume two major folding motifs in various media: (i) the single stranded β6.3 helical dimer (“channel” form) and (ii) the double stranded intertwined helix (collectively known as the “nonchannel” form) (see Figure 7A). The head-to-head (amino terminal-to-amino terminal) single-stranded β6.3 helical dimer form is the cation conducting channel conformation of gramicidin in membranes. In this conformation, the carboxy terminus is exposed to the membrane
water interface, and the amino terminus is buried in the hydrophobic core of the membrane. This places the tryptophan residues clustered at the membrane
water interface at the entrance to the channel.114,115,128 The channel interior is lined by the polar carbonyl and amide moieties of the peptide backbone, a feature shared with the selectivity filter of the bacterial KcsA potassium channel.129 An important aspect of

the channel conformation is the membrane interfacial location of the aromatic residues (tryptophan), a common feature of many transmembrane helices (see Figure 7A).95,114,115,128 We previously showed that the tryptophan residues of gramicidin in the channel conformation exhibit REES, implying that the tryptophan residues are localized in the interfacial region and experience motional restriction.114,115 This preferential localization of aromatic amino acids such as tryptophan is shared by many membrane proteins and is believed to serve as a driving force to stabilize functional membrane-bound conformations leading to switches between “on” and “off” states.100 An important application of REES is to monitor the conformational transition of membrane proteins. For example, REES has been conveniently used to explore conformational changes of membrane-bound gramicidin (see Figure 7B).115 This is due to the fact that while the tryptophan residues are clustered toward the membrane interface in the channel conformation of gramicidin they are distributed along the membrane axis in the nonchannel conformation. This implies that the environment of the tryptophans in these cases is very different, giving rise to differences in REES (7 nm for the channel conformation compared to 2 nm for the nonchannel conformation).115 In addition, we monitored REES displayed by intermediates in the folding pathway of membrane-bound gramicidin from the initial 5701

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The Journal of Physical Chemistry B nonchannel to the final channel conformation (denoted as intermediates I and II). Interestingly, intermediates I and II display REES of 3 and 4 nm, respectively. The progressive increase in REES from the nonchannel conformation of gramicidin (2 nm) to the channel conformation (7 nm) via the intermediate conformations (3
4 nm) correlates well with the gradual conversion of the nonchannel form to the channel form. These results demonstrate that REES can be conveniently employed as a tool to monitor membrane protein folding. The tryptophan residues in gramicidin channels are crucial for maintaining the structure and function of the channel.127 The importance of the tryptophans has been demonstrated by the observation that the cation conductivity of the channel decreases upon substitution of one or all of the tryptophan residues by phenylalanine, tyrosine, or naphthylalanine and also upon ultraviolet irradiation or chemical modification of the tryptophan side chains. In a recent study, we explored the structural basis for the reduction in channel-forming propensity using single tryptophan analogues of gramicidin utilizing REES and other fluorescence approaches.111 The advantage of using single tryptophan analogues is that ground state heterogeneity in the tryptophan environment is avoided, and any information obtained could be correlated to the unique tryptophan residue in the sequence. Our results showed that the single tryptophan analogues adopt a predominantly nonchannel conformation in membranes.

5. CONCLUSION AND FUTURE PERSPECTIVES In this article, we have focused on the application of REES in exploring membrane organization and dynamics. A general concern in the case of fluorescence studies in motionally restricted systems is that red edge effects could complicate results if fluorophores are excited at the red edge of the absorption spectra rather than at the absorption maxima. Studies such as energy transfer could therefore lead to significant errors unless carried out with caution. In fact, it has been previously reported that there is a wavelength-dependent variation in the measured location (depth) of fatty acyl attached probes in the membrane.72 A potentially exciting application is to obtain a detailed dynamic and topological map of membrane proteins and peptides using a combination of site-specific mutation and labeling.130 This assumes relevance in view of recent reports, based on crystal structures of membrane proteins such as rhodopsin and β2-adrenergic receptor, that there are motionally restricted water molecules that could be important in inducing conformational transitions in the transmembrane portion of the receptor.131 Water plays a crucial role in the formation and maintenance of proteins and membranes.43,44 Knowledge of dynamics of hydration at a molecular level is therefore of considerable importance in understanding the membrane structure and function. REES is based on the change in fluorophore
solvent interactions in the ground and excited states induced by a change in the dipole moment of the fluorophore upon excitation and the relative rate at which solvent molecules reorient around the excited state fluorophore. Since in the case of membranes the ubiquitous solvent is water, the information obtained in such cases will have its origin from the otherwise optically silent water molecules. The unique feature of REES is that while other fluorescence measurements (such as fluorescence quenching, energy transfer, anisotropy measurements) yield information about the fluorophore itself REES provides information about the relative rates of solvent

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(water in the case of membranes) relaxation (reorientation) dynamics that is not possible to obtain by other approaches. This makes the use of REES particularly useful in membrane research since hydration plays a crucial modulatory role in a large number of important cellular events involving the membrane such as lipid
protein interactions and ion transport.132,133

’ AUTHOR INFORMATION Corresponding Author

*Phone: þ91-40-2719-2578. Fax: þ91-40-2716-0311. E-mail: [email protected].

’ BIOGRAPHIES

Sourav Haldar received his B.Sc. from the Department of Chemistry, Jadavpur University, Kolkata, in Chemistry (Honors) in 2003 and M.Sc. from the Department of Chemistry, Indian Institute of Technology, Madras (Chennai), in 2005. He is presently pursuing his Ph.D. at the Centre for Cellular and Molecular Biology, Hyderabad. His major interest is in fluorescence spectroscopy, membrane organization, dynamics, and lipid
protein interactions.

Arunima Chaudhuri received her B.Sc. degree in Physiology (Honors) from Presidency College, University of Calcutta, in 2005. She then received her M.Sc. degree in Marine Biotechnology from Goa University in 2007. She is currently a graduate student at the Centre for Cellular and Molecular Biology in Hyderabad. Her research includes monitoring lipid
protein interactions in biological membranes and membrane
mimetic systems such as micelles and reverse micelles using fluorescence as a major technique. 5702

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Prof. Amitabha Chattopadhyay received B.Sc. with Honors in Chemistry from St. Xavier’s College (Calcutta) and M.Sc. in Chemistry from Indian Institute of Technology, Kanpur. He obtained his Ph.D. from the State University of New York at Stony Brook and was a Postdoctoral Fellow at the University of California, Davis. He subsequently joined the Centre for Cellular and Molecular Biology (CCMB) in Hyderabad, where he is currently an Associate Director. Prof. Chattopadhyay’s work is focused on monitoring organization, dynamics, and function of biological membranes utilizing fluorescence-based spectroscopic and microscopic approaches. His other major interest is the role of membrane lipids in the organization and function of G-protein coupled receptors. Prof. Chattopadhyay was awarded the prestigious Shanti Swarup Bhatnagar Award by the Prime Minister of India. He is an elected Fellow of all the Indian Academies of Science.

’ ACKNOWLEDGMENT Work in A.C.’s laboratory was supported by the Council of Scientific and Industrial Research and Department of Science and Technology, Government of India. S.H. and Ar.C. thank the Council of Scientific and Industrial Research for the award of Senior Research Fellowships. A.C. is an Adjunct Professor at the Special Centre for Molecular Medicine of Jawaharlal Nehru University (New Delhi, India) and Indian Institute of Science Education and Research (Mohali, India) and Honorary Professor of the Jawaharlal Nehru Centre for Advanced Scientific Research (Bangalore, India). A.C. gratefully acknowledges J.C. Bose Fellowship (Department of Science and Technology, Government of India). Some of the work described in this article was carried out by former members of A.C.’s group whose contributions are gratefully acknowledged. We thank members of our laboratory for critically reading the manuscript. ’ REFERENCES (1) Granick, S. Motions and relaxations of confined liquids. Science 1991, 253, 1374–1379. (2) (a) Bhattacharyya, K.; Bagchi, B. Slow dynamics of constrained water in complex geometries. J. Phys. Chem. A 2000, 104, 10603–10613. (b) Jha, A.; Ishii, K.; Udgaonkar, J. B.; Tahara, T.; Krishnamoorthy, G. Exploration of the correlation between solvation dynamics and internal dynamics of a protein. Biochemistry 2011, 50, 397–408. (3) Levinger, N. E. Water in confinement. Science 2002, 298, 1722–1723. (4) Mentr
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