Archives of Biochemistry and Biophysics 580 (2015) 102–111

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Review

Electron spin resonance of spin-labeled lipid assemblies and proteins Rita Guzzi, Rosa Bartucci ⇑ Department of Physics, University of Calabria, 87036 Rende (CS), Italy

a r t i c l e

i n f o

Article history: Received 1 April 2015 and in revised form 18 June 2015 Accepted 22 June 2015 Available online 24 June 2015 Keywords: Spin-label ESR Membrane Protein Rotational dynamics Polarity

a b s t r a c t Spin-label electron spin resonance (ESR) spectroscopy is a valuable means to study molecular mobility and interactions in biological systems. This paper deals with conventional, continuous wave ESR of nitroxide spin-labels at 9-GHz providing an introduction to the basic principles of the technique and applications to self-assembled lipid aggregates and proteins. Emphasis is given to segmental lipid chain order and rotational dynamics of lipid structures, environmental polarity of membranes and proteins, structure and conformational dynamics of proteins. Ó 2015 Elsevier Inc. All rights reserved.

1. Introduction Electron spin resonance (ESR) is a spectroscopic technique that detects the resonant absorption of microwave radiation by a substance with at least one unpaired electron spin placed in a static magnetic field. It is widely used in different fields from basic research to industrial applications [1]. For systems that are ESR active, the technique is suitable to study the structure of solid and liquid samples and is useful to investigate dynamic processes. In biophysics, ESR represents a valid tool to characterize transition metal complexes, active site of metalloproteins, and radicals formed under a variety of conditions [1–4]. Its field of application has been extended to diamagnetic biosystems, such as lipid membranes, proteins, nucleic acids, by using the spin-labeling technique [5–7]. A spin-label is a stable free radical that can be introduced at a specific site in biological macromolecules and allows ESR to be applied. Spin-labels and spin-labeled molecules can be synthesized and are also commercially available. Versatile species are those based on the nitroxide –NO radical [5,6,8]. Advances in molecular biology concerning protein mutagenesis has determined a strong impulse in ESR spectroscopy through site-directed spin labeling (SDSL) methodology. In SDSL any protein residue can be selectively substituted with cysteines for spin-label attachment [9–11]. Spin-label ESR is commonly used at 9 GHz (X-band), but spectrometers working at lower frequency (1 GHz, L-band; ⇑ Corresponding author. E-mail address: rosa.bartucci@fis.unical.it (R. Bartucci). http://dx.doi.org/10.1016/j.abb.2015.06.015 0003-9861/Ó 2015 Elsevier Inc. All rights reserved.

3 GHz, S-band) or higher frequency (24 GHz, K-band; 35 GHz, Q-band; 94 GHz, W-band; 130 GHz, D-band; 250 GHz, J-band) are also available. They are designed to operate in the continuous wave (cw), in the linear and non-linear regime under progressive saturation conditions, or saturation transfer. Some spectrometers operate in the pulsed Fourier Transform (FT) mode and in the time-resolved domain. Spin-label ESR spectroscopy is largely driven by the sensitivity of the label to its surroundings. The motional sensitivity of the technique extends on a timescale range from 1012 to 106 s (i.e., T2 spin–spin relaxation timescale) to 106–103 s (i.e., T1 spin–lattice relaxation timescale). This timescale matches the one characteristic of molecular motions of lipid and protein components in membranes. The length-scale range goes from ca. 5–80 Å, important for structural investigations of biomacromolecules. Intra- and intermolecular distance determinations are possible; the distances sampled in singly labeled cw- and FT-ESR are increased by using double labeling and pulsed double ESR techniques [12–15]. X-band ESR of nitroxide spin-labels holds a prominent place among the spectroscopic techniques for the investigation of the structure and function of enzymes and proteins, slow translational motions of lipids and proteins (lateral lipid and protein diffusion in membranes, anisotropic protein rotations) and fast anisotropic lipid rotations (long axis rotations, angular motions, chain segmental motions), lipid/protein interactions, and local polarity of membrane and protein regions [5–7,14,16–19]. For the improved orientational resolution and motional sensitivity on a faster timescale, high field/high frequency spin-label ESR gives insights into nonaxial rotations and both lateral and transverse ordering of the

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phospholipid chains in membranes, rapid internal motions of proteins, and environmental polarity [20–26]. The librational dynamics at cryogenic temperatures and the direct detection of solvent accessibility to specific biosystem sites [27–36] are addressed by electron spin echo-based methods of pulsed FT-ESR [37]. An overview of different ESR methods applied to several biological systems can be found in [38,39]. In this work the basic principles underlying the conventional, cw-ESR spectroscopy of nitroxide spin labels at 9 GHz are presented along with applications to self-assembled lipid aggregates and proteins.

2. Nitroxide spin-labels Most biological systems do not possess an intrinsic paramagnetism which is necessary for ESR spectroscopy. The technique is therefore applied by using a stable free-radical or ‘‘spin-label’’, i.e., an external paramagnetic probe which contain one unpaired electron spin residing in a molecular orbital. The spin-label can be inserted in the sample and acts as a reporter group. The corresponding ESR signal originates exclusively from the label site, so that site-specific, ensemble-averaged molecular and dynamical information can be obtained [5,6,8]. The most common used spin-label moieties are based on the nitroxide radical, –NO, where the unpaired electron spin is localized on the 2pp orbital of the nitrogen atom, 14N. The –NO group is flanked by quaternary carbon atoms, which protect the radical

103

and account for the stability of the label. The –NO group is enclosed either in a six-membered piperidine (TEMPO), or a five-membered pyrroline/pyrrolidine (proxyl) rings, or in an oxazolidinyl (doxyl) ring (Fig. 1a–d) [5,6,8,14]. For ESR studies of lipid aggregates, lipid membranes, and lipid/protein complexes appropriate spin-labels are the spin-labeled lipids, i.e., lipids to which the spin label group can be attached to the polarhead or is rigidly and stereospecifically bound to selected carbon atom position, C-n, along the hydrocarbon chain [5,6,19]. In Fig. 1e–g a polar-head labeled lipid and chain-labeled lipids are shown as an example. They are TEMPO-stearate, i.e., a stearic acid molecule in which the piperidine ring is on the polarhead, 5-stearic acid spin label (5-SASL), i.e., a stearic acid molecule in which the doxyl group is on the 5th carbon atom of the acyl chain, and 14-phosphatidylcholine spin-label (14-PCSL), i.e., a di-palmitoylphosphatidylcholine molecule bearing the nitroxide moiety on the 14th carbon atom of the sn-2 chain. To avoid spin–spin interaction, the spin-labeled lipids are introduced at very low concentration (typically 0.5–1 mol% of the total lipid concentration) in the basic lipid matrix. They are considered endogenous lipids that mix well with the parent lipids with the polarheads in register among them and the hydrocarbon tails in contact. For ESR studies of proteins, spin-labels able to react towards specific groups of amino acid residues by forming a covalent bond are used. The features of the spin-labels depend on the functional group attached to the nitroxide ring. Maleimido and iodoacetamide spin-labels alkylate both sulphydryl and amino groups, MTSSL binds

Fig. 1. Nitroxide radicals: (a) six-membered piperidine ring; (b) unsaturated five-membered pyrrolidine ring; (c) saturated pyrroline ring; (d) doxyl ring. Spin-labeled lipids: (e) 2,2,6,6-tetramethyl-piperidin-1-oxyl-4-yl octadecanoate, TEMPO-stearate; (f) 5-(4,4-dimethyloxazolidinyl-N-oxyl)stearic acid, 5-stearic acid spin label, 5-SASL; (g) 1acyl-2-(14-(4,4-dimethyloxazolidinyl-N-oxyl)stearoyl)-sn-glycero-3-phosphocholine, 14-phosphatidylcholine spin label, 14-PCSL. Spin-labels used in covalent modification of proteins: (h) 4-maleimido-2,2,6,6-tetramethylpiperidine-1-oxyl, 6-MSL; (i) maleimide spin-label 3-maleimido-tetramethylpyrrolidine-1-oxyl, 5-MSL; (j) 4-(2Iodoacetamido)-2,2,6,6-tetramethylpiperidine 1-oxyl; (k) 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin label, MTSSL.

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selectively to free sulfhydryl group on cysteines (see Fig. 1h–k). An ESR spectrum of a spin-labeled protein contains contributions from (i) the rotational motion of the entire protein or peptide, (ii) fluctuations of the a-carbon backbone, and (iii) the residual motion of the label relative to the peptide backbone [5,14]. Depending on the experimental conditions, these motions can be resolved from the spectrum as they lie on different timescale to which ESR spectroscopy is sensitive. A development in the spin-labeling technique is achieved with SDSL [9–11,38]. It is based on site-directed mutagenesis of a generic residue to cysteine followed by modification of the sulfhydryl group with a nitroxide reagent to generate a stable ESR active side chain. To this end, one of the most commonly used spin label is MTSSL for its sulfhydryl specificity and small volume. For selective SDSL, it is desirable to have only one cysteine residue in the sample. When the target protein contains additional native nondisulfide bonded cysteines these should be replaced with residues non-reactive to MTSSL (for example, alanine). With site-directed mutagenesis, the nitroxide spin-labels can be introduced at almost any selected site within a protein sequence. In this way extended protein regions can be investigated at high spatial resolution, a map of protein motions can be drawn out and the protein structure and conformational dynamics detailed characterized [40–43]. 3. Basic principles of electron spin resonance and spectra of nitroxide spin-labels The lineshape of an ESR spectrum of a nitroxide spin-label can be described by the following spin Hamiltonian [3,5,17]:

H ¼ bH  g  S þ I  A  S

ð1Þ

The first term represents the Zeeman interaction between the unpaired electron spin S = 1/2 and the static magnetic field H and b is the Bohr magneton; the second term is the hyperfine interaction between the electron spin and the nuclear spin I = 1 of the 14N atom of the –NO nitroxide moiety. The two interactions are anisotropic as indicated by the g-factor tensor, g, and the hyperfine tensor, A. This makes the ESR spectra critically dependent on the orientation of the spin-label with respect to the magnetic field. To the nitroxide group is associated a Cartesian molecule-fixed axes system in which the x-axis is directed along the –NO bond and the z-axis is along the 2pp orbital where the unpaired spin is localized. The orientation of the magnetic field H is given by the polar angles h and u (see Fig. 2). It is generally assumed that the molecular axes system coincides with both the principal-axes system of the g and A tensor. Thus, g and A are simultaneously both diagonal in this molecular axes system. Under such conditions Eq. (1) becomes:

and the energy levels are given by:

The electron spin quantum number ms takes the values 1=2 and the nitrogen nuclear spin quantum number mI can take the values 1, 0, and 1. The order of magnitude of the energy corresponding to the Zeeman interaction is greater than that of the hyperfine interaction and the effect of this latter is to split each of the two Zeeman ms levels in three levels, one for each mI values = 0, ±1 (see energy levels in Fig. 2). In an ESR experiment an oscillating magnetic field 2H1 cos 2ptt, where H1 is circularly polarized rf field component, is applied perpendicular to H, inducing transition between the spin states, provided that the frequency m satisfies the resonance conditions DE ¼ hm. The spectrum is therefore given by:

hv ¼ g zz bHmI þ Azz mI

ð5Þ

where h is Planck’s constant. According to the selection rules DmS ¼ 1 and DmI ¼ 0, three resonant transitions are allowed which are centered at H0 ¼ hv =g zz b and have equal hyperfine splittings: H0  H1 ¼ Azz =g zz b (see Fig. 2). This is the basic principle of electron spin resonance. For a static magnetic field of about 3300 Gauss, it is required a radiation with a frequency ca. 9 GHz, i.e., in the microwave region of the electromagnetic spectrum. A similar analysis can be made for H oriented along the x and y axes of the spin label, when the nitroxide molecules are dissolved in single-crystal hosts. From Fig. 2 it can be seen that, because the components of the g and A tensors are different, the ESR spectra depend strongly on the orientation of the magnetic field H. For a generic orientation of the magnetic field, specified by the polar coordinates h and u relative to the nitroxide z-axis, the ESR transition are: ðh;uÞ

hv ¼ gðh; uÞbHmI

þ Aðh; uÞmI

ð2Þ

ð3Þ

ð6Þ

where 2

2

gðh; uÞ ¼ ðg xx cos2 u þ g yy sin uÞ sin h þ g zz cos2 h

ð7Þ

and 2

2

Aðh; uÞ ¼ ½ðA2xx cos2 u þ A2yy sin uÞ sin h þ A2zz cos2 h

1=2

ð8Þ

The hyperfine tensor A is to a good approximation axial: Axx  Ayy  A? (6 Gauss)  Azz (32 Gauss), and a small anisotropy is seen (at 9 GHz) in the g-value: gxx > gyy > gzz. In the case of axial symmetry, the diagonal elements of g and A are: 2

where gii and Aii ði ¼ x; y; zÞ are the principal g-values and hyperfine tensor elements, respectively, Hi are the components of the static magnetic field, Si and Ii the components of the electron and nuclear spin, respectively, with respect to the molecular axes. The energy levels and the ESR spectroscopic transitions are obtained by solving the spin Hamiltonian depicted in Eq. (2). For nitroxides in single crystal hosts, the magnetic field can be oriented along each of the principal axes x, y, and z and from the corresponding spectra the principal tensor elements gii and Aii can be obtained. As an example, if H is oriented along the z molecular axis, the spin Hamiltonian Eq. (2) reduces to:

H ¼ bg zz HSz þ Azz Iz Sz

ð4Þ

gðhÞ ¼ g ? sin h þ g k cos2 h

H ¼ bðg xx Hx Sx þ g yy Hy Sy þ g zz Hz Sz Þ þ Axx Ix Sx þ Ayy Iy Sy þ Azz Iz Sz

E ¼ bg zz Hms þ Azz mI ms

2

AðhÞ ¼ ðA2? sin h þ A2k cos2 hÞ

ð9Þ 1=2

ð10Þ

For nitroxides that are not oriented in single crystals but consist of a random distribution of orientations of the molecular axes, the resulting spin-label ESR spectrum is the superposition of spectra from the three principal axes and all intermediate orientations. Since the ESR spectra of spin labels are dependent upon the molecular orientation, reorientation by molecular motion will affect the spectra. Molecular motion can be characterized by a rotational correlation time sc , that, for a sphere of radius r undergoing isotropic motion in a medium with viscosity g; is given by the Stokes– Einstein relation sc ¼ 4pgr3 =3kT. The conventional ESR timescale extends from 1012 s to 106 s, optimal motional sensitivity is on the nanosecond regime and it is determined by the transverse spin–spin relaxation time, T2 [5,6,17,18].

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Fig. 2. (Left) Energy levels and allowed ESR transitions for the nitroxide system, S = 1/2 and I = 1. (Right) Molecular axes system associated with the nitroxide –NO group. Simulated single crystal nitroxide spectra with the magnetic field oriented along each of the principal axes x, y and z. Immobilized and isotropic spectra are also shown. Spectra are simulated by using EasySpin [44].

If the nitroxide motions are appreciably slower than the conventional ESR timescale with rotational correlation time sc P 10ns, the so-called rigid limit, powder spectrum is obtained (see Fig. 3). It is due to the rigidly-immobilized randomly-oriented spin-labels in a viscous solvent or at low temperature. In the rigid limit spectrum only the outer extrema of the hyperfine splitting, 2Amax , can be measured, whereas the inner hyperfine component, 2Amin , merge with the central hyperfine line and cannot be resolved. When the motions are fast on the conventional ESR timescale with sc 6 1011 s an isotropic spectrum is obtained (see Fig. 3). It arises from spin-labels rapidly- and randomly-tumbling in a non-viscous solvent or at high temperature. The fast isotropic motion averages the spin-Hamiltonian terms depending on orientation (i.e., the g-values and A-tensor anisotropy) and a narrow, three-lines spectrum is obtained. In this case, the isotropic g-value, g 0 , and the isotropic hyperfine splitting, A0 , are then given by:

g 0 ¼ ð1=3Þðg xx þ g yy þ g zz Þ

ð11Þ

A0 ¼ ð1=3ÞðAxx þ Ayy þ Azz Þ

ð12Þ

In the case of axial symmetry, they are given by:

g 0 ¼ ð1=3Þð2g ? þ g k Þ

ð13Þ

A0 ¼ ð1=3Þð2A? þ Ak Þ

ð14Þ

Considering the isotropic spectrum, as the motion progressively slows, the spectral lineshape (line positions, linewidths and line splittings) changes (see Fig. 3). The effect of motional restriction is evident first in a differential line broadening while the line positions remain constant. Then a distortion of line position and lineshapes is observed until the rigid powder spectrum is reached for conventional ESR. In the slow motional regime, however, saturation transfer ST-ESR results in spectra still sensitive to molecular motion down to millisecond. i.e., spin–lattice relaxation time, T1 [6,17,18]. Spin-label ESR spectra are acquired by varying linearly the magnetic field while keeping constant the microwave frequency and supplying the microwave power well below saturation. Continuous-wave 9 GHz ESR spectrometers use field modulation normally at 100 kHz and phase sensitive detection that results in the ESR spectra being displayed as the first derivative with respect

Fig. 3. Simulated conventional ESR spectra of nitroxide spin-labels with different rotational correlation time. Simulation are performed by using EasySpin [44].

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to the magnetic field scan. The spectra are centered at ca. 3300 Gauss (in the g = 2 region) and require a sweep width of ca. 100–120 Gauss. Spectral parameters, such as hyperfine splittings and linewidths, can be measured from the spectra and used to determine the order parameter and the rotational correlation time of a spin-label as described elsewhere [5,6]. Numerical simulations, possibly combined with least-squares fitting, are powerful tools to reproduce the magnetic signal and resolve the underlying structural and dynamic parameters. A comprehensive software package, able to simulate ESR spectra of different paramagnetic systems under a wide range of dynamic conditions, is EasySpin [44]. Other simulation programs are described in [4,17,24]. 4. Applications of spin-label ESR in lipid structures and proteins 4.1. Segmental chain order and rotational dynamics of self-assembled lipid structures The transverse chain ordering and the segmental rotational dynamics of self-assembled lipid aggregates such as bilayer lamellar and micellar phases are conveniently studied with cw ESR at 9 GHz by using chain labeled lipids. In Fig. 4 are shown the ESR spectra at 10 °C of 5- and 16-PCSL in bilayers of DPPC, in interdigitated bilayers formed by DPPC in the presence of mercury chloride HgCl2 (lipid/ions molar ratio = 1:2), and in micelles of the polymer-lipid PEG:2000-DPPE. Saturated symmetrical chain phosphatidylcholines, of which DPPC is the most studied lipid, in water self-assemble in bilayers. The lipid bilayer is the basic structural arrangement of biomembranes and represent a highly dynamical template by virtue of

Fig. 4. ESR spectra of 5-PCSL (upper spectra of each pair, black lines) and 16-PCSL (lower spectra of each pair, red lines) in bilayers of DPPC (upper), interdigitated bilayers of DPPC + HgCl2 (middle), and micelles of PEG:2000-DPPE polymer-lipid (lower). T = 10 °C and spectral width = 100 Gauss. A schematic representation of state of aggregation of lipid in water are also shown.

the rotational motion of the lipid chain segments [45]. The spectrum at 10 °C of 5-PCSL in DPPC bilayers (Fig. 4a) shows a high degree of anisotropy with 2Amax ca. 65 Gauss. It is typical of immobilized spin label in the ordered gel phase Lb0 , in which the lipid chains are in the all-trans configuration and tilted relative to the bilayer normal. At the same temperature, the spectrum of 16-PCSL (Fig. 4b) displays a considerable lower anisotropy and 2Amax is reduced to ca. 47 Gauss, indicating that same motion in the slow motional regime of the conventional ESR timescale is occurring in the inner hydrocarbon region. 2Amax is a useful empirical measure of chain dynamics and ordering valid in both slow and fast motional regimes of the conventional nitroxide ESR timescale [5,6]. Large values reflect a high degree of spin-label immobilization. The spectral difference between the two extreme positions of chain labeling is typical of bilayer lamellar assemblies in the gel phase, where the motion increases and the order decreases on moving along the lipid chain from the first segments (probed by 5-PCSL) to the bilayer midplane (probed by 16-PCSL) [46]. It has been found that under various conditions the opposing DPPC monolayers may interpenetrate each other to form the interdigitated gel phase LbI, in which the terminal methyl groups of the lipid chains are located near the interfacial region on the opposite side of the lipid lamella (Fig. 4, middle) (for a review, see [47]). Indeed, above a certain threshold concentration, compounds such as glycerol, small surface-active molecules, alcohols, ions, and proteins are able to induce complete interdigitation in phosphatidylcholines at temperatures corresponding to the gel state. Chain interdigitation is absent for temperatures in the fluid phase where normal noninterdigitated bilayers are formed. In the LbI phase, which is characterized by the loss of the bilayer midplane, the motion of an end-label, such as 14-, 16-PCSL or 14-, 16-SASL, is restricted to an extent similar to that experienced by a label probing the region adjacent the glycerol backbone, in proximity of the polar/apolar interface (5-, 7-PCSL or 5-, 7-SASL) [46,48–50]. In other words, in the interdigitated phase the spectrum of an end-label shows a larger anisotropy with respect to the spectrum of the same label in conventional noninterdigitated bilayer phases. This is clearly seen by comparing the spectra of 16-PCSL in the interdigitated phase of DPPC induced by HgCl2 (Fig. 4d) and in bilayers of DPPC (Fig. 4b). Moreover, the difference D2Amax (for n-SASL or n-PCSL) between two extreme positions of labeling (n = 5 and 16) is 18 Gauss for conventional DPPC bilayers and it notably reduces in lamellae with interdigitated chains. In fact, D2Amax is very low for interdigitated bilayers of DPPC in glycerol and in ethylene glycol (1.3 and 3.1 Gauss, respectively). In other interdigitated bilayers, such as those induced by methyl mercury, chaotropic ions and ethanol, it varies between ca. 7 and 10 Gauss, respectively [46,49,50]. The D2Amax values account for the different chain packing density (loosened packed vs. more compact structure) among the various interdigitated phases. Double-chained polymer-lipids of dipalmitoylphosphatidyletha nolamine derivatized on the polar head with the bulky polymer poly(ethylene glycol) of molecular mass of 2000 Da (PEG:2000-DPPE) adopt micellar structures when dispersed in water, as occurs for single-chained lysolipids. PEG-lipids are essential components of sterically stabilized ‘‘Stealth’’ liposomes used in drug-delivery and micelles of polymer-lipids can vehicle hydrophobic drugs as well [51]. The spectra at 10 °C of 5- and 16-PCSL in micelles of PEG-2000-DPPE (Fig. 4e and f) show a marked lower spectral anisotropy when compared to the corresponding spectra recorded in lamellar bilayer structures of DPPC. They indicates that the labels are in a fluid environment already at low temperatures. The observed variation in the spectral anisotropy reflects a decrease of the lipid chain packing density on going from bilayer membranes of diacyl lipids to large and complex polymer–lipid micelles.

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On the whole, the spin-label ESR spectra in Fig. 4 clearly highlight the difference in chain segmental mobility that results from the different modes of chain packing in the various lipid assemblies (normal and interdigitated lamellar phases and micelles). Further insight into molecular properties of lipid aggregates can be obtained from the positional dependence of the spectral anisotropy at high temperature. The ESR spectra of n-PCSL in DPPC bilayers in the fluid phase at 50 °C (Fig. 5, black lines) are typical of labels in the fast motional regime and display a continuous averaging of the spectral anisotropy on moving along the chain from the first segments adjacent to the polar/apolar interface (5-, 7-PCSL) down to the terminal methyl end of the chain (14-, 16-PCSL). The spectra collapse to isotropic triplets with differential line broadening for spin labels close to the terminal methyl end. This behavior indicates a gradient of increasing rotational mobility on proceeding toward the DPPC bilayer midplane. The flexibility profile, also observed, although to a lesser extent, in the gel phase, is a hallmark of lamellar fluid lipid assemblies. It has been observed for phospholipids bilayers, biological membranes, and lipid/protein complexes [5–7,46,52–54]. The fundamental molecular process that gives rise to membrane mobility or fluidity is the progressive trans-gauche rotational isomerisms about the C–C bonds of the chains. As it can be seen from the ESR spectra of n-PCSL in Fig. 5, a flexibility gradient along the hydrocarbon chain also exists in micellar assemblies of PEG:2000-DPPE (Fig. 5, red lines). A striking feature in Fig. 5 is that at the polar/apolar interface the spectral anisotropy is appreciably lower in micelles of polymer–lipids than in bilayers of DPPC, and this difference reduces markedly towards the methyl end of the chains. This contrasts with small micelles in which conformational intrachain disorder is higher than that in the liquid crystalline phase of diacyl-PC with the same chain length on the whole length of the chain [52].

4.2. Environmental polarity An interesting application of spin-label ESR is related to the polarity determination of the environment in proximity of the nitroxide. The accessibility of solvent across the hydrocarbon region of membranes, or the hydrophobicity at specific sites of biomolecules, or the variation of polarity along selected protein regions can be studied [14,55,56]. This is because the dielectric constant and the H-bonding propensity of solvent influence the Zeeman interaction (i.e. g-values) and the hyperfine interaction (i.e., Azz and A0 ). The three spectral parameters, g xx , Azz and A0 , reflect environmental polarity and proticity around the spin-label via the dependence on the net unpaired electron spin density on the nitroxide –NO reporter group. In general, an increase of polarity or hydrogen bonding in the vicinity of the spin-label group will favor a greater spin density on the nitrogen atom, and this leads to an increase in the hyperfine couplings and to a decrease of the g-value. At 9-GHz, determinations of the Azz component of the hyperfine tensor are made at low temperature, in the frozen state when the rotational dynamics is absent. The spin-label ESR spectra are in the rigid limit of motional sensitivity of conventional spin-label ESR and Azz is equal to half of the outer hyperfine splitting, measured from the spectra. At higher frequencies (i.e., 95 GHz), this parameter can be evaluated directly from the spectra for the enhanced Zeeman resolution of rigid-limit spectra of disordered samples [57]. Determinations of the 14N-hyperfine isotropic coupling value, A0 , are made in the fluid state at a sufficiently high temperature, where the rotational dynamics does not influence the spectral lineshape. A0 is evaluated from the hyperfine splittings according to Eq. (14). The highest sensitivity of g-values to polarity is attained for the g xx tensor element at high-frequency, high-field 95-GHz measurements [57,58]. Polarhead-labeled and chain-labeled lipids are used to delineate, at high spatial resolution, the transmembrane polarity profiles by plotting one of the ESR observable (i.e., g xx , Azz , A0 ) vs. n. Fig. 6 gives the dependence of A0 on the position, n, of chain labeling for the n-PCSL in aqueous bilayer dispersions of DPPC and DPPC with equimolar cholesterol [59]. The data in Fig. 6 show a trough-like transmembrane polarity profile and are fitted by the Boltzmann sigmoidal function:

A0 ðnÞ ¼

Fig. 5. ESR spectra at 50 °C of different positional isomers of n-PCSL in fluid bilayers of DPPC (black lines) and in micelles of PEG:2000-DPPE (red lines). Spectral width = 100 Gauss. Adapted from Ref. [52].

107

A0;1  A0;2 þ A0;2 1 þ eðnn0 Þ=k

ð15Þ

Fig. 6. Positional dependence of the isotropic 14N-hyperfine coupling constant, A0, of n-PCSL in fluid bilayer membranes of DPPC and of DPPC + 50 mol% cholesterol. Adapted from Ref. [59].

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where A0;1 and A0;2 are the limiting values of A0 at the polar headgroup and terminal methyl ends of the spin-label chain, respectively, k is an exponential decay constant, and n0 specifies the midpoint of the transition. The trend clearly indicates that, within the hydrocarbon region of the bilayer membranes, a transition takes place from a high-polarity region ðn < n0 Þ adjacent to the lipid headgroups (probed with 5- and 7-PCSL) to a low-polarity region ðn > n0 Þ at the bilayer midplane in the center of the membrane (probed with 14- and 16-PCSL). The characteristics of the transition, i.e., amplitude, determined by A0;1 and A0;2 , width, determined by k, and position, determined by n0 , all depend on lipid composition. In membranes containing equimolar cholesterol (Fig. 6) the width is left unaffected, the point of steepest slope is shifted to higher n0 , and the amplitude of the transition is increased for the shift to higher polarities closer to the membrane surface and to lower polarity at the membrane mid-plane. These changes are likely associated with the ordering effect of cholesterol on fluid lipid chains in addition to a spacing effect at the polar head group region of the lipids. Qualitatively, similar effects of cholesterol have been observed on the polarity profiles delineated by Azz in frozen membranes [60], g xx at high frequency [57], and on the transmembrane water penetration profiles determined by measuring the positional dependence of the intensity of the solvent (deuterium) in the Fourier Transform-ESR Electron Spin Echo Envelope Modulation (ESEEM) spectra [30]. These latter determinations confirmed that the hydrophobic barrier in bilayer membranes results from the reduced accessibility of the lipid membrane interior to water. The results shown in Fig. 6 describe a general behavior: a hydrophobic barrier with a sigmoidal shape is observed in model membranes of different composition, in natural membranes and in membranes of their extracted lipids [30,33,56,57,60–62]. Polarity data are also used to obtain structural and topological information on membrane proteins and to reveal information on protein fold. Azz vs. g xx plots from high-field ESR on site-directed spin labeled transmembrane protein bacteriorhodopsin disclosed sites with aprotic character typical of the cytoplasmatic moiety of the proton channel and sites typical of the protic environment in the extracellular channel [58,63]. Minimum polarity (low Azz , high g xx ) is found in the middle of the proton channel between the proton donor D96 and the retinal chromophore. A periodical polarity pattern of alternation of polar and apolar residues are evidenced in the alpha-helical structure of the sequence 88-94 in the first HAMP domain of the natronomonas pharaonis halobacterial transducer II, in complex with sensory rhodopsin, [64]. In a combined cw- and pulsed X-band ESR study on site-directed spin labeled light-harvesting complex of photosystem II of green plants (consisting of a membrane protein and several cofactors) the Azz values from the protein samples have been compared with those from reference measurements on a free spin probe in various solvents with different dielectric constant [34]. The study identifies a group of residues with large Azz in environments less polar than a water/glycerol mixture (e = 64) but more polar than methanol (e = 32.6), a group with intermediate Azz in an environment with a polarity comparable to that of methanol, and a group with low Azz with polarity similar to that of isopropanol (e = 18). In addition, for a residue close to the N-terminus, in a domain unresolved in X-ray structures, the results indicate high solvent accessibility.

4.3. Spin-labeled proteins: structure, dynamics and conformational transitions Spin-label ESR is widely used to explore the structure, dynamics and conformational substates of soluble globular proteins as well as membrane proteins. It is also applied to study protein–ligand

interaction in which the label is either on the protein or on the ligand [40,65–69]. Protein domains which are not defined in the crystal structure are successfully investigated by SDSL–ESR. An interesting example is the 100 amino-acid residue long N-terminus of the plant antenna membrane protein CP29 labeled at 55 positions [70]. Multicomponent spectra are recorded and their features resolved by spectral simulations. The single-components are grouped in three mobility categories from slow (with sc > 50 ns) to medium (with sc  4 ns) to fast component (with sc  1 ns). Topographic regions that differ considerably in their dynamics, identified by using Hubbell’s plots [71,72], are: loop/surface (highest mobility region), loop/contact, helix/surface, helix/contact and helix/buried sites (lowest mobility region). Residues close to the transmembrane part of the protein gradually transit from flexible to helix surface to helix buried region. Moreover, ESR spectroscopy in combination with SDSL has notably contributed to study intrinsically disordered proteins, i.e., proteins which are mostly unfolded in their native state and in the absence of binding partners. An example is alpha-synuclein (aSN), a water-soluble presynaptic protein (140 residues, 14.4 kDa) with a pathological role in Parkinson’s disease. The protein becomes stabilized in an amphiphatic alpha-helical structure upon interacting with anionic membranes in the fluid phase. Monomeric aSN labeled through the sequence gives rise to sharp ESR line shapes that are characteristic for loop or unfolded regions [73,74]. In the presence of lipid membranes, little or no spectral changes are observed at positions within the C-terminal region of the polypeptide chain. In contrast, changes are detected for labels in the N-terminal domain (1–100 residues) and all the spectra exhibit lineshapes characteristic of lipid- or solvent-exposed helix surface sites. The results clearly indicate that the C-terminal is an unstructured, flexible protein region, whereas the N-terminal region folds on interacting with membranes. ESR, both cw and pulsed, combined with molecular dynamics simulation refinements, are used to assign local secondary protein structure and further contribute to determine the membrane immersion depth of lipid-exposed residues and to provide molecular insight into the mechanism by which aSN interacts with membranes [73–76]. In a number of proteins, the access to the active sites is controlled by a surface loop or lid which undergoes a transition between two different conformations: open vs. closed. Such dynamic process is found to occur in human pancreatic lipase, as observed in crystallography. To monitor this conformational change in solution in the presence of amphiphiles and lipid substrate, a paramagnetic probe has been inserted in the lid by selective site mutation [77]. Two-component ESR spectra with different dynamical features are recorded and assigned to the closed and open conformations of the lid. Protein conformational substates and the timescale of exchange between them can be detected from ESR spectroscopy. For spin-labeled T4 lysozyme, conventional X-band cw-ESR is combined with pulsed saturation recovery experiments to sample an extended timescale region and to distinguish and, in some cases, separate different nitroxide environments: fast rotameric exchange are found to lie in the 0.1–1 ls range, whereas slow conformational exchange is at least an order of magnitude slower [78,79]. By using singly spin-labeled mutants of myoglobin distributed throughout the sequence, the flexibility profile of protein regions has been mapped: fast backbone fluctuations and regions in slow conformational exchange in folded and partially folded protein sequences have been evidenced [80]. Exchange between conformational substates has been identified and resolved in complex membrane proteins such as vitamin B12 transporter [43]. Insight into structural, dynamical and molecular processes occurring in proteins are gained by studies at cryogenic

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temperatures. In the following we present applications aimed to detect protein conformational substates and characterize librational fluctuations. They are observed at low temperatures and can drive functionally important motions at physiological temperatures. The energy landscape of proteins is usually described by a hierarchy of conformational substates, which give rise to a structural heterogeneity at low temperatures [81]. A dynamical transition takes place at temperatures in the region of 200 K, above which the proteins then fluctuate between different conformational substates. Existence of protein conformational substates has been well established by a number of experimental techniques, including X-ray crystallography [82], Mössbauer spectroscopy [83], and neutron scattering [84,85]. For metalloproteins in the frozen state, g- and A-strain in ESR spectra have been related to structural disorder and conformational substates [4,86,87]. Recently, heterogeneity of protein substates have been visualized by cw and spin-echo ESR methods in multiply spin-labeled large transmembrane protein Na, K-ATPase [88,89], and in smaller, singly-labeled water-soluble proteins of different secondary structure [90]. These latter include the two a-helical proteins human hemoglobin (Hb) and HSA, and the bLG, a b-sheet protein. The proteins were spin-labeled on a single cysteine residue by using a maleimido nitroxide derivative (Fig. 1h), and were examined in water, in the freeze-dried state, and in a glass-forming 60% v/v glycerol-water mixture. The cw-ESR spectra are immobilized powder patterns over a wide temperature range (120–240 K) and have rather broad lines, which narrow progressively with increasing temperature (see Fig. 7). Evidences of protein conformational substates are obtained from line-shape analysis of the low-field ðmI ¼ þ1Þ hyperfine line of each ESR spectrum. The line has been fitted by non-linear least-squares minimization with a Voigt absorption line, i.e., a Gaussian convolution of pure Lorentzian components, as follows:

v ðHÞ ¼ A

Z

þ1

2

expððH0  H0 Þ =2r2G Þ

dH

0

ð16Þ

109

A heterogeneous population of conformational substates is identified by inhomogeneous broadening (i.e., pronounced Gaussian broadening, high DHG ) at low temperatures. A dynamical transition to a uniform population characterized by homogeneous line broadening (low DHG ) was found at higher temperatures, above 200 K [90]. This signature is particularly significant for proteins in water/glycerol mixture. Most likely, the glassy solvent promotes substate heterogeneity in the proteins and points to solvent-mediated protein transitions. Several dynamically nonequivalent sites were observed in the X- and D-band ESR spectra of MTSSL spin-labeled photosynthetic bacterial reaction center, which are believed to reflect distinct conformational substates of the protein structure [91]. The motions that drive transitions between the different protein conformational substates are identified in librations, i.e., rapid oscillations in the nanosecond timescale of small amplitude (< 15 ) [92,93]. Evidence of the librational motion are obtained from the motionally relaxed echo-detected ED-ESR spectra. The characteristics of the motion (i.e., mean-square amplitude and rotational correlation time) are then determined by combining pulsed and cw-ESR data [29,31]. Librational fluctuations whose amplitude and/or frequency depend on temperature and solution conditions were observed by linear (linewidth measurements at low, DHþ1 , and high, DH1 , field) and saturation transfer cw-ESR for the 6-MAL spin-label immobilized in hemoglobin in the temperature range 120–41 °C [94,95]. Analysis of the multifrequency ESR spectra of the spin-labeled reaction center protein describes the dynamics as fast librations in a cone with a correlation time faster than 109 s [91]. The mean-square amplitude of librational motion, ha2i, can be derived from the motionally averaged hyperfine splitting, hAzzi, in the cw-ESR spectra of spin-labeled proteins, according to [96]:

ha2 i ¼

Azz  hAzz i Azz  Axx

ð17Þ

where DHL is the half-width at half-height of each Lorentzian component, rG is the standard deviation of the Gaussian distribution of Lorentzian components, and H0 is the center of the Gaussian distribution. The width at half-height of the Gaussian distribution is pffiffiffiffiffiffiffiffiffiffiffiffi given by DHG ¼ ð2 2 ln 2ÞrG .

where Axx , Ayy and Azz are the principal elements of the hyperfine tensor. Fig. 8 shows the temperature dependence of the libration amplitude for three spin-labeled proteins, Hb, HSA and bLG in water and in 60% v/v glycerol-water mixture. Interestingly, the ha2i temperature dependence that is recorded by ESR resembles that of the mean-square displacement,

Fig. 7. Temperature dependence of the cw-EPR spectra of 5-MSL-labeled bLG in a 60% v/v glycerol–water mixture. Crosses represent fitting of the low-field ðmI ¼ þ1Þ hyperfine extremum with a Voigt lineshape, Eq. (16). Total scan width = 100 G.

Fig. 8. Temperature dependence of the mean-square amplitude of librational motion, ha2 i, for spin labeled proteins in water (upper panel) and in 60% v/v glycerol-water mixtures. Solid lines are fits with Eq. (18). Adapted from Ref. [97].

1

2

ðDHL =2Þ2 þ ðH  H0 Þ

110

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hr2i, measured by Mössbauer spectroscopy and quasi-elastic neutron scattering [98,99]. Librational motions of appreciable amplitude set in above 200–210 K, for the proteins in water and slightly above this temperature for the proteins in glycerol–water mixtures. Thus, onset of librational oscillations coincides with the protein dynamical transition. Moreover, the variation between the librational amplitudes of the different systems is an effect of solvent viscosity (and also of the structural diversity of the different single sites of labeling) and reflects coupling of dynamic processes to the environment. To this last point, it is worthy to note that librations are properties of the hydrated proteins, because they are absent for the lyophilized spin-labeled proteins [35,95,100]. The temperature dependence of the mean-square amplitude of librational motion in Fig. 8 has been described by the Vogel–Tam man–Fulcher equation that is appropriate for the relaxation processes in glass-forming solvents [101]:

ha2 i ¼ A expðBT 0 =ðT  T 0 ÞÞ

ð18Þ

where T 0 is the temperature at which a2 ! 0 and A and B are fitting parameters. The fits are adequate to establish that T 0 increases when 60% glycerol is added to the medium. For T T 0 , above the dynamic transition, Eq. (18) simplifies to: ha2 i  expðBT 0 =TÞ and ha2i vs. 1=T can be approximated by the Arrhenius law with activation energies of about 20 kJ/mol in water and about 30–40 kJ/mol in glycerol. These values are in the range of those estimated from the mean-square displacement hr2i and relaxation processes in glass-forming media and the hydration shells of proteins [85,101,102]. Thus, rapid librational motion becomes appreciable at the temperatures required for crossing the activation barriers that separate conformational substates. Earlier ESR studies on carbonmonoxy hemoglobin [95], myoglobin and lysozyme [103], and on methaemoglobin [100], each spin-labeled with 6-MSL, have found similar low-temperature behavior to that reported for oxyHb, bLG and HSA. In each case, a discontinuity in temperature dependence of ESR spectral parameters, such as the outer hyperfine splitting, the width at half-height and the order parameter, is found at around 200–210 K for the hydrated proteins, but not for the lyophilized ones. 5. Conclusions In this review applications of conventional 9 GHz ESR spectroscopy of nitroxide spin-labels in lipid assemblies and proteins are described. It is evidenced the sensitivity of the technique to nanosecond rotational motions and to the surroundings around the label. ESR spectra of spin-labels in lipid matrices display lineshapes whose anisotropy depend on temperature (i.e., physical state of the lipids) and on the host lipid structure (i.e., bilayers, interdigitated bilayer or micelles). Transmembrane flexibility and polarity profiles are delineated by using lipid spin probes with the nitroxide systematically stepped down the hydrocarbon chain. Structural, dynamical and topological information on proteins are gained by SDSL–ESR. Conformational substates and librational dynamics of spin-labeled proteins are visualized and characterized by lineshape analysis and hyperfine splitting determination of low temperature EPR spectra. The present review shows that spin-label ESR is a valuable tool to address biochemical and biophysical aspects of biosystems under many different experimental conditions. Transparency Document The Transparency document associated with this article can be found in the online version.

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