Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy.

Volume 16 Number 21 7 June 2014 Pages 9669–10234

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Themed issue: 15th Anniversary Issue ISSN 1463-9076

PERSPECTIVE Daniella Goldfarb Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy

Published on 06 December 2013. Downloaded by State University of New York at Stony Brook on 30/10/2014 18:58:10.

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Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy† Daniella Goldfarb Methods for measuring nanometer scale distances between specific sites in biomolecules (proteins and nucleic acids) and their complexes are essential for describing and analyzing their structure and function. In the last decade pulse EPR techniques were proven very effective for measuring distances between two spin labels attached to a biomolecule. The most commonly used spin labels for such measurements are nitroxide stable radicals. Recently, a new family of spin labels, based on Gd3+ chelates, has been introduced to overcome some of the limitations of using nitroxides, particularly at high magnetic fields, which are attractive due to the increased sensitivity they offer. The benefits that such S = 7/2 spin labels offer for frequencies of 30 GHz and higher, particularly at 95 GHz, include (1) high sensitivity, only B0.15 nmol of doubly labeled biomolecule is needed, (2) the lack of orientation selection, which allows straightforward data analysis. Gd3+–Gd3+ DEER (double electron–electron resonance) distance measurements on labeled peptides, proteins and DNA have already been demonstrated and the results show that they are very promising in terms of sensitivity. In this Perspective we review these new developments. We briefly introduce the characteristics of the DEER experiment on a pair of S = 1/2 spins and characterize the EPR spectroscopic properties of Gd3+ ions. We then introduce some of the tags employed to attach Gd3+ to biomolecules and provide a few experimental examples of Gd3+–Gd3+

Received 8th September 2013, Accepted 6th December 2013

DEER measurements. This is followed by a discussion of the parameters that affect the sensitivity of such

DOI: 10.1039/c3cp53822b

(ZFS), its effect on the DEER modulation frequencies must be considered and this is discussed next. Finally, another recently reported approach for using Gd3+ in distance measurements will be presented:

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the use of Gd3+–nitroxide pairs.

DEER measurements. Since an important term in the spin Hamiltonian of Gd3+ is the zero-field splitting

1. Introduction Electron-paramagnetic resonance (EPR) techniques are highly effective for determining the distances between strategic sites in biological macromolecules such as proteins, nucleic acids, and their assemblies.1 A collection of such distance measurements can, for example, provide sparse structural information that can be used for tracking conformation changes upon ligand/substrate binding in solution, or can be used as constraints for modeling, and can determine how individual protein subunits with known structures are assembled in larger structures. At the heart of this methodology lies the availability of well defined paramagnetic probes, between which the distances are measured. These could be either intrinsic paramagnetic centers such as paramagnetic transition metal ions2 and radicals3 or artificially introduced spin labels.4 Up to a few years ago the

Department of Chemical Physics, Weizmann Institute of Science, Rehovot 76100, Israel. E-mail: [email protected] † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c3cp53822b

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field of spin labeling was dominated by nitroxide stable radicals,4 which were first introduced by McConnell in the 1960s.5 Their EPR spectrum, in frozen solutions, can reveal dipolar interactions between nitroxides situated 0.7–2.0 nm apart.6 This range can be extended to 8.0 nm by applying pulse EPR methods.1,7,8 These methods, often referred to as pulsed dipolar spectroscopy (PDS), measure the dipolar interaction between two electron spins, which is readily converted into a distance due to its r3 dependence, where r is the interspin distance. Nitroxide spin labels are usually attached to proteins by site-directed spin labeling (SDSL), where the spin label is conjugated through thiol groups of native or mutated cysteine residues.9 Recently, the incorporation of an unnatural amino acid to which a nitroxide can be attached has been reported.10 Methods have also been devised for labeling nucleic acids with nitroxides.11 Distance measurements carried out on commercial X-band (B9.5 GHz, B0.35 T) spectrometers, using nitroxide spin labels, are nowadays practically routine. This is due to the establishment of experimental techniques, particularly the deadtime-free fourpulse DEER (double electron–electron resonance, often also called PELDOR (pulse electron double resonance)) sequence

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Fig. 1 (a) The four-pulse DEER sequence.12 (b) A schematic representation of the W-band EPR spectrum of an S = 1/2 spin pair with a frequency difference of Do and a dipolar coupling of odd.

(see Fig. 1a),12 and the availability of data analysis algorithms and software for extracting the distance distribution from the data.13–15 The major drawback of using X-band DEER is its rather limited sensitivity, which is particularly critical for biological samples. The sample for X-band DEER requires 30–80 ml of B0.1 mM solution of the doubly labeled biomolecule, and the measurement time is about 12–24 h at 50 K.16 This varies according to the distance, and accessing distances above 6 nm is significantly more challenging. Several approaches are currently being pursued for increasing the sensitivity of DEER distance measurement and broadening its scope of applications. These include the use of perdeuterated proteins,17 application of shaped pulses,18,19 and increasing the spectrometer frequency to Q-band (32 GHz, 1.2 T), where nitroxide spin labels still perform very well.20,21 Further increasing the spectrometer frequency to W-band (95 GHz, B3.5 T) would also increase the sensitivity, provided that the mw (microwave) power is sufficient to produce short enough pulses.22,23 W-band DEER measurements require only 2–3 ml of B50 mM protein solutions23 or B200 ml of 1 mM solutions,22 depending on the spectrometer configuration. A caveat in using this approach is that the resolved nitroxide g-anisotropy leads to orientation selection24,25 and the extraction of distance distributions from DEER data requires a series of measurements at several magnetic field positions along the EPR powder pattern.26–31 This is also associated with a rather complex data analysis because, in addition to the distance distribution, 5 additional angular parameters have to be determined.

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Nonetheless, when the labels are rigid, this analysis provides additional structural constraints and currently efforts are being made to automate such data analysis.32–34 The above-mentioned limitations prompted the introduction of new spin labels for DEER measurements at high magnetic fields. These are based on Gd3+, which is a half integer, high-spin ion (S = 7/2) with half-filled valence f orbitals that provides high sensitivity at high magnetic fields. More importantly, it is free from orientation selection effects in DEER.35 Lanthanide tags are routinely used in paramagnetic NMR, where they produce pseudo-contact shifts (PCS) or paramagnetic relaxation enhancements (PRE), and the necessary chemistry for conjugating such tags to proteins has been developed.36 These tags have also been used for PRE generation in EPR in conjunction with nitroxide spin labels.37,38 Since the first report of Gd3+–Gd3+ DEER on a model compound in 2007,35 there have been a number of reports on distance measurements between pairs of Gd3+ ions in model systems,39 peptides,40,41 proteins,42,43 nucleic acids,44 and coated nanoparticles.45 These reports show that such spin labels make an attractive alternative to nitroxide for distance measurement applications at spectrometer frequencies from Q-band and above. In this Perspective, we review the approach of Gd3+ spin labeling for distance measurements. We start with a brief introduction of the DEER experiment on a pair of S = 1/2 spins (Section 2), and then describe the EPR spectroscopic properties of Gd3+ ions that make them attractive spin labels for DEER distance measurements (Section 3). In Section 4 we present some tags employed to attach Gd3+ to biomolecules and give a few experimental examples of Gd3+–Gd3+ DEER measurements. Next, we discuss the various parameters that affect the sensitivity of such measurements and how they are controlled experimentally (Section 5). In Section 6 we discuss the effects of the zero field splitting (ZFS) on the observed DEER modulation frequencies. Finally, in Section 7 we present another approach for employing Gd3+ in distance measurements: the use of Gd3+–nitroxide pairs. We end with an outlook on future possibilities and suggestions for further developments needed to fully realize the potential of Gd3+ spin labeling for distance measurements by EPR methods.

2. DEER measurements A schematic EPR spectrum of two coupled S = 1/2 spins, A and B, having sufficiently different resonance frequencies and a dipolar coupling of odd, is depicted in Fig. 1b, where odd = o0dd(3 cos2 y 1)

(1)

2

m0 gA gB ðbÞ and y is the angle between the 4p hr3 ! inter-spin vector, r, and the external magnetic field, B 0 , gA and gB are the g factors of the two spins and b is the Bohr magneton. In the DEER experiment (Fig. 1a) a spin echo is generated on spin A by microwave pulses on resonance with nA = nobs, whereas spin B is selectively inverted by a p pulse on resonance with nB = npump. The application of the p pump pulse on spin B In eqn (1) o0dd ¼


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leads to a change of DMS(B) = 1, thus shifting the resonance frequency of spin A by the dipolar splitting, |odd|, when the frequency separation Dn = |nobs npump| > odd/2p. This frequency shift leads to dephasing, which cannot be refocused by the echo formation. The time variation, t, of the pump pulse thus generates oscillations in the echo intensity, according to24,46 ð1 ðp Vintra ðtÞ ¼ 1 l PðrÞ ½1 cosðodd tÞdr sin y dy (2) 0

0

In eqn (2) P(r) is the intra-pair distance distribution and l, referred to as the modulation depth parameter, is the probability of flipping spin B. In an orientationally disordered system, like a pair of nitroxide spin labels in a frozen solution, where all ! orientations of the pair with respect to B 0 are excited by the pulses, l is independent of y. Eqn (2) holds for odd/2p { |nA nB| and |odd/2p| { |nA|,|nB|. These conditions are commonly met for X-band DEER measurements on a nitroxide pair. In addition, the conditions o1 c odd and o1/2p { |nA nB| are required for eqn (2) to hold. o1 is the amplitude of the pump pulse. It is o1 that determines the low-end range of the distances accessible by DEER. The distance distribution is usually extracted by solving the inverse of eqn (2) using the DeerAnalysis software package.13 The intermolecular dipolar interaction between two labels on different molecules also contributes to the DEER trace. It is manifested as an exponential decay in isotropic solutions and is usually removed during data analysis.7 This holds for cases where the spin lattice relaxation time T1 c (t + t1) and spin flips owing to spectral diffusion can be neglected. The sensitivity of the DEER experiment depends, in addition to spectrometer-specific characteristics, on both l and the echo intensity at tmax = (t + t1) according to1,7,46,47 2tmax pffiffiffiffiffiffi1 T1 SðDEERÞ / lV exp (3) TM In eqn (3) V is the echo intensity for tmax = 0; we neglected the effect of the intermolecular dipolar interaction that adds to the exponential decay. Furthermore, we assumed that the echo decay is exponential. T1 is taken into account because it affects the efficiency of data averaging. The modulation depth, l, under orientation selection-free conditions, is a function of the pump pulse amplitude, o1, frequency, and duration, tp, and the EPR lineshape, g(Do), according to48 ð1 2 o1 2 O tp gðDoÞdðDoÞ l¼ sin (4) 2 2 0 O O2 = o12 + Do2. The off-resonance frequency, Do, is given with respect to the pump pulse frequency. The value of l can be determined through the asymptotic (1 l) value of Vintra(t) (see eqn (2)). For nitroxide spin labels and X-band measurements l B 0.3–0.4 and V is a function of g(Do) and of the observer pulses’ frequency and duration. Eqn (4) does not take into account the limited bandwidth of the resonator and it holds for cases where the pulse bandwidth is lower than the resonator bandwidth and the


EPR spectrum. This usually applies for pump pulses longer than 12 ns applied to nitroxides. According to eqn (3), large l, large V, long TM, and short enough T1 values are desired. The optimum measurement temperature for nitroxides, 50 K, is determined by TM and T1.7 For a Gd3+ pair, both l and V are temperature dependent because the lineshape is temperature dependent at high fields and low temperatures.2 Accordingly, one has to understand the EPR spectroscopic characteristics of Gd3+ (S = 7/2) in order to appreciate its utility as a spin label for determining distances by DEER. This is briefly reviewed in the next section.

3. The Gd3+ EPR spectrum Gd3+ has 7 unpaired electrons in its f orbitals, resulting in a 8S (S = 7/2) ground state. Two isotopes of Gd, 155Gd and 157Gd, with a natural abundance of B15% each, have a nuclear spin of 3/2 and a small hyperfine coupling on the order of B16 MHz.49 This small coupling is not resolved in the EPR spectrum of a frozen solution and can be neglected. Accordingly, the spin Hamiltonian of a Gd3+ pair is i X h ! hSÂ T S^B H¼ gbe B0 S^zi þ hSî Di Sî þ (5) i¼A;B

The first term in eqn (5) corresponds to the isotropic electron spin Zeeman interaction ( g B 2,49 depending on the spectrometer frequency, due to second order shift by the ZFS). The second term is the zero-field splitting (ZFS) term, with principal components Dxx,i, Dyy,i and Dzz,i (given in frequency units) where conventionally |Dxx,i| o |Dyy,i| o |Dzz,i|. Di is usually traceless and is characterized by two values, Di = 3/2Dzz,i and Ei = (Dxx,i Dyy,i)/2. For simplicity we will assume axial symmetry, where Ei = 0. Usually the two Gd3+ tags are the same and therefore their principal D values are the same. The third term in the Hamiltonian is the dipolar coupling between the two Gd3+ ions. Because this interaction is relatively weak and usually is not resolved in the EPR spectrum, for simplicity we can neglect this term when discussing the EPR lineshape and we can drop the index i. The first-order EPR transition frequencies derived from this spin Hamiltonian are

1 2MS þ 1 oMS !MS þ1 ¼ gbe B0 þ D 3 cos2 b 1 ; h 2

(6)

ˆZ operator and b is the angle where MS is the projection of the S ! between B 0 and the direction of Dzz. To first order, the central transition, |1/2i - |1/2i, is independent of D. There is, however, a second order contribution given by50 ð2Þ

o1=2!1=2 ¼

D2 h ð4SðS þ 1Þ 3Þ½2ðsin2 2bÞ þ ðsin4 bÞ 16gbe B0 (7)

4 hD2 1 the central transition gbe B0 remains narrow and therefore it confers high sensitivity to Gd3+ at high fields. The width of the other transitions is field independent and is determined by eqn (6). Eqn (7) shows that as long as


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The allowed EPR transition probabilities are given, to first order, by


PMS-MS+1 = [S(S + 1) MS(MS + 1)]

(8)

This yields transition probabilities of (7, 12, 15, 16, 15, 12, 7) for the Gd3+ seven transitions, whereas for S = 1/2 the transition probability is unity. This has important consequences in terms of the microwave power (MW) required for achieving short pump pulses in the DEER measurements. The MW nutation frequency for a given transition is o1;ðMS !MS þ1Þ ¼

gb 1=2 B1 ; P h ðMS !MS þ1Þ

(9)

where B1 is the amplitude of the MW irradiation. Thus, the same power will generate a p pulse about 4 times shorter when applied to the Gd3+ central transition, as compared with that applied to a S = 1/2 system. This efficient use of MW power for high spin systems is crucial for high field measurements, where the availability of MW power is limited. In addition, this allows producing short pulses, which together with the broad Gd3+ spectrum could, in principle, access short distances, usually inaccessible with DEER on nitroxides.51 A unique feature of Gd3+ chelates in frozen solutions is the large distributions of D and E and of the orientation of the principal axis system of the ZFS tensor with respect to the molecular frame.52,53 This leads to smearing of the powder pattern singularities of the ‘‘other’’ (except central) D-dependent transitions. It is this feature that is responsible for the lack of orientation selection in Gd3+–Gd3+ DEER. At W-band the EPR spectrum comprises a narrow line due to the central transition, superimposed on a broad featureless background, as shown in Fig. 2a for Gd3+–DOTA at 10 K. Fig. 2b presents a calculated 10 K W-band spectrum obtained with D and E values and distributions typical for Gd3+–DOTA in frozen solutions. The figure also displays sub-spectra of the individual EPR transitions. The low-temperature EPR spectrum of Gd3+ is not only spectrometer frequency dependent but is also temperature dependent, particularly at high frequencies where thermal polarization becomes substantial. At low temperatures, the lower energy levels become more populated at the expense of the higher energy levels and this consequently changes the

Fig. 3 (a) The energy level diagram for Gd3+ at 95 GHz and at a low temperature. The thickness of the lines represents relative populations. (b) The Boltzmann population difference of the central transition, |1/2i |1/2i, at W (95 GHz)- and Q (34 GHz)-bands.

relative intensities of the various transitions in the EPR spectrum, as seen in Fig. 2b. In Fig. 3 we compare the temperature dependence of the population difference of the central transition of Gd3+ at Q- and W-band. These plots show that a maximum intensity of the central transition is reached at B10 K and B3 K at W- and Q-band, respectively.

4. Gd3+–Gd3+ distance measurements In principle, because of the narrow central transition of Gd3+ at high magnetic fields, it should be possible to resolve dipolar interactions already in the continuous wave (CW) EPR spectrum. A first step towards this new direction has been recently reported at 240 GHz (8.6 T), where the residual linewidth of the central transition of GdCl3 in a frozen solution is 0.5 mT.55 This linewidth was measured in the concentration range of 0.1 mM to 50 mM, covering a large range of average inter-spin distances. The spectra exhibited substantial dipolar broadening at average distances up to B3.8 nm, which is significantly longer than the limit for nitroxides at X-band. Clear line broadening could also be observed in a 260 K CW EPR spectrum, offering Gd3+ spin labels for temperatures higher than possible with conventional pulsed EPR distance measurements.55

Fig. 2 (a) W-band echo-detected EPR spectrum (10 K) of Gd3+–DOTA. The inset zooms in on the central transition. The positions of the observer and pump frequencies in DEER measurements are denoted by arrows. (b) Calculated (using EasySpin)54 W-band EPR spectrum of Gd3+ at 10 K. The individual transitions are shown as well and are magnified in the inset. The parameters used are D = 600 MHz with a Gaussian distribution of |D|/2, E/D in the range of 0–1/3 with a probability P(E) = E/D 2(E/D)2.47



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For DEER measurements on Gd3+–Gd3+ pairs to be efficient and easily applied, they should behave similar to a S = 1/2 pair, where data analysis is straightforward and the modulation frequency is free of any contributions from the ZFS. In analogy to a pair of S = 1/2 spins, at a particular orientation with respect to the magnetic field (Fig. 1b), the spectrum of a S = 7/2 pair will exhibit, to first order, a total of 14 sets of transitions, each split into a multiplet of 8 lines (octate) with a splitting of odd. Let us consider the following situation: a particular multiplet, |MS(A), MS(B)i - |MS(A) + 1, MS(B)i, of spin A is observed, and another non-overlapping transition multiplet of spin B, |MS(A), MS(B)i - |MS(A), MS(B) + 1i, is pumped. The pump pulse generates a change of DMS(B) = 1 and this causes a frequency shift of some of the multiplet components of A spins by |odd|, just as in the case of an S = 1/2 pair. Such a situation, where different spins are affected by the pump and observer pulses, is highly probable considering the large inhomogeneous broadening of the Gd3+ spectrum. A detailed discussion of this assumption can be found in Section 6. The first Gd3+–Gd3+ DEER measurements were carried out at Q- and W-band frequencies on a rigid bis-Gd3+ model compound, shown in Fig. 4a.35 The pump pulse was set to the maximum of the central transition to achieve the largest l value, and the observer pulse was set to the edge of the central transition. The W-band DEER trace, recorded at 25 K, along with the distance distribution extracted using DeerAnalysis13 is shown in Fig. 4b and c. The maximum of the distance distribution, at 2.03 nm, agrees well with the distance of 2.12 nm obtained later from the crystal structure.56 Some notable points are the relatively low modulation depth, B4%, and more importantly, the relatively broad distance distribution, considering the rigidity of the model compound. This is addressed in Section 6. Next, Gd3+–Gd3+ DEER measurements were demonstrated on a flexible model, with an

Fig. 4 (a) The structure of the bis-Gd3+ model compound. (b) The fourpulse DEER trace of the model compound shown in (a) recorded at W-band, 25 K (durations of observed pulses were t0 = 16/32/32 ns, duration of pump pulse was tp = 16 ns, Dn = 83 MHz). (c) The corresponding distance distribution.35


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average short distance and a broad distance distribution, again both at Q- and W-band frequencies.39 DEER measurements were also reported on proteins42,43 and peptides40,41 where the Gd3+ chelate was attached by adapting methods from paramagnetic NMR and SDSL, as shown in Fig. 5. The Gd3+ chelates used for DNA labeling44 are also shown in Fig. 5 and here the attachment is based on click chemistry. Table 1 lists the width of the central transition of some of these tags at W-band, along with the corresponding estimated D value (center of distribution). In Gd3+–Gd3+ DEER measurements it is desirable not to have free Gd3+ in solution since it would contribute to the background decay. This is particularly critical for tags that have a larger D value than that of the aqua Gd3+ complex, like 4MMDPA, where the contribution of the free Gd3+ at the maximum of the overlapping central transitions is higher than its actual relative concentration.40,42 In principle, chelates with the highest possible binding constants are needed to avoid leakage of Gd3+ to other sites in the biomolecule. Furthermore, such chelates can be attached to the protein with the bound Gd3+, thereby avoiding the presence of free Gd3+. Finally, a small label is preferred, in order to disturb as little as possible the protein structure. In this context, the 4MMDPA and its analogs are attractive, although their binding constant is lower than that of DOTA.40,41 The C1 tag is rather large, designed to be attached to the external surface of the protein, and its bulky substituents confer rigidity to the tag.58 A comparison of the size of the MTSSL tag, used in the SDSL labeling of proteins, and that of the Gd3+–DOTA tag is presented in Fig. S1 in the ESI.† In the first report on Gd3+–Gd3+ DEER on proteins,42 two proteins with known NMR structures, P75 and t14, were labeled at the same position with a pair of Gd3+–4MMDPA tags or nitroxide spin labels. In both cases the nitroxide–nitroxide distance, determined from X-band DEER, was somewhat shorter than the Gd3+–Gd3+ distance. Modeling based on the NMR structures revealed a good agreement with the experimentally determined Gd3+–Gd3+ distance distribution for both proteins, whereas the nitroxide–nitroxide distance distributions were underestimated in the modeling. Here all the available conformational space of the label was sampled and conformations positioning heavy atoms within 0.15 nm of each other were rejected. While for the Gd3+ this was sufficient, for the nitroxide this approach does not take into account its hydrophobic bias.41 Nitroxide– nitroxide DEER measurements were carried out at W-band as well. The W-band P75 results were similar to those of the X-band, thus revealing no orientation selection owing to flexibility. In contrast, for t14 clear orientation selection was observed, which prevented a straightforward extraction of the distance distribution.42 Examples of DEER results on a doubly Gd3+-labeled protein with the C1 tag43 and a double-stranded DNA44 with the Gd-538 tag are shown in Fig. 6 and 7, respectively. As shown, Gd3+–Gd3+ distances of 6 nm are readily accessed at both Q- and W-band measurements. The length of the DEER evolution time of the W-band measurements (tmax B 11 ms) indicates that distances of 8 nm should be accessible as well. In all cases, the obtained distance distribution agreed very well with either the crystal/NMR structures or those obtained by molecular dynamics (MD) simulations.


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Fig. 5 Tags used for Gd3+–Gd3+ distance measurements: (a) Gd3+–DOTA (DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) tag attached via a cysteine residue.57 (b) A DOTA derivative with bulky substituents, Gd3+–C1, attached via cysteine residues.58 (c) and (d) Gd3+-595 and Gd3+-538 attached to DNA via click chemistry.44 (e) Gd3+–4MMDPA (4-mercaptomethyl-dipicolinic acid), also attached via cysteines.36

Table 1 List of the Gd3+ compounds and the width of their central transitions (FWHH) at W-band, along with their D values (center of the distribution)

Chelate 3+

Gd –DOTA Gd3+–aquo complex Gd3+-538 Gd3+-595

DH (mT) 1.3 2.7 3.4 1.25

0.1 0.2 0.15 0.05

D (mT) B2053 B3040 B4038 B2038

Recently, Gd3+–Gd3+ distance measurements were demonstrated in a membrane environment.41 Fig. 8 shows W-band DEER traces of two WALP23 peptides (Ac-CWWLALALALALALALALALWWCNH2), labeled with two C1 (WALP23-C1) or DOTA (WALP23-DOTA) tags at both ends of the peptides, inserted into phospholipid multi-lamellar vesicles. The corresponding distance distributions are shown in Fig. 8b. Whereas the maximum of the distance distribution is at 3.7 nm for WALP23-C1, it is longer, 4.2 nm, for WALP23-DOTA. This difference was attributed to a conformational bias due to the preferred interaction of the label with the membrane; the C1 being positively charged is pushed more into the membrane, towards the phosphate region.41 The hydrophobic substituents can also contribute to this bias. Calculated Gd3+–Gd3+ distance distributions obtained for a perfect a-helix and for all possible conformations of the tag, not in a membrane, generated similar distance distributions for the two tags, supporting the above interpretation of the experimental difference. Measurements were also carried out on WALP19, 21, 25, and 27 peptides, again


with Gd3+–C1 tags at their ends. The discrepancy between the maximum of the calculated distance and those distances determined experimentally in the membrane remained throughout the series. Remarkably, the distances observed were in accordance with the increased length of the helix and exhibited a cis–trans effect following the orientation of the cysteine residues with respect to the helix axes.41 These sets of measurements are very encouraging because they indicate that Gd3+–Gd3+ DEER measurements on membrane proteins with multiple helices, using minute amounts of protein (>0.15 nmol) and acceptable measurement times (several hours), are feasible. Finally, Mn2+ (S = 5/2) has spectroscopic properties similar to Gd3+, except for the presence of a 55Mn hyperfine interaction. Accordingly, W-band Mn2+–Mn2+ DEER distance measurements were also reported on a protein with two attached Mn2+ tags.59 Most of the reported Gd3+–Gd3+ DEER measurements were carried out at 10–25 K with the pump pulse set at the central transition and the observer pulses set at 70–100 MHz away, either at the edge of the central transition or on the broad background, depending on the width of the central transition. This experimental set-up yielded a modulation depth, l, that was rather shallow and did not exceed 5% (see Table 2 in ref. 2). Q-band measurements with particularly short pump pulses of 12 and 8 ns reached l values of 9%. In the next section, we discuss the optimum experimental conditions for achieving the best DEER sensitivity and the largest l value.


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Fig. 8 W-band Gd3+–Gd3+ DEER results obtained for WALP23/DOPC.41 (a) Background-corrected normalized W-band DEER traces (black traces) for WALP23-C1 and WALP23-DOTA tags. The trace of the WALP23-DOTA experiment was shifted up by 0.03 units. The red lines represent the fit with the distance distributions shown in (b). (b) Distance distributions (black lines) obtained using DeerAnalysis for WALP23-C1 and WALP23-DOTA. Blue lines denote modelled distance distributions.

5. Sensitivity considerations

Fig. 6 W-band DEER results obtained with about 3 mL of 100 mM frozen solutions of the ERp29 mutant S114C in 80% D2O/20% glycerol-d8 (v/v) at 10 K.43 (a) Normalized DEER traces fitted with appropriate background decay (in red). (b) The same DEER trace after background removal along with the fits obtained either by Tikhonov regularization (red) or by fitting two Gaussians (blue). (c) Distance distribution obtained by the two different fits shown in (b). The data were analyzed using the program DeerAnalysis.13

Fig. 7 (a) Experimental (10 K) Ka-band (solid) and calculated (dashed) DEER trace after background removal of double-stranded Gd3+-538-DNA. The calculated trace is based on the distribution function shown in the inset, dashed line. Inset: dashed line, distance distribution function, P(r), obtained with a single Gaussian fit using DeerAnalysis (the maximum is at 5.4 nm); the solid line, P(r) obtained by MD simulations. Reprinted from ref. 44, Copyright 1990, with permission from Elsevier. (b) W-band DEER trace (10 K, after background subtraction) of the same sample as in (a). The red trace is the calculated trace obtained from the distance distribution given in the inset, obtained with two Gaussian fits using DeerAnalysis. The maximum is at 5.7 nm.


A recent publication47 was devoted to optimizing Gd3+–Gd3+ DEER measurements and to comparing Q- and W-band measurements. When optimizing DEER SNR on a pair of Gd3+ spin labels using a particular instrument and a cavity, one has to consider the parameters of eqn (3). We begin with the relaxation times, and as an example, we present temperature-dependent relaxation data on 0.05 mM Gd3+–DOTA in a D2O/glycerol-d8 (7 : 3, v/v) solution. The glycerol is needed for creating a good glass with welldispersed solutes, and the deuteration extends the phase memory time. Measurements were carried out at the maximum of the central transition and at 10 mT (280 MHz) higher. In most of the samples, the echo decay deviated slightly from the exponential decay, but since the deviation was small, we decided to continue to use this as a reasonable approximation. The results obtained in the range of 6–25 K, displayed in Fig. 9a, reveal that (i) TM is temperature dependent and that (ii) TM of the central transition is consistently longer than that measured on the background due to the other transitions. This suggests that the observer pulse should be set to the central transition and that lower temperatures are preferred. Saturationrecovery measurements yielded a recovery curve that could be well fitted with a biexponential function, with slow and fast time constants. We assumed that the slow time constant, T1s, represents the spin lattice relaxation and the results are shown in Fig. 9b. At the moment we do not know the origin of the fast component. T1s increases as the temperature decreases and, unlike TM, no significant difference was found between the two field positions. In Fig. 9c we have plotted the product 2tmax T10:5 for tmax = 5 ms, showing that the relaxation exp TM behaviour suggests an optimal temperature of 10–15 K. For shorter tmax values, 25 K is a better choice, although here random flips, arising from the faster component of the magnetization recovery, have a destructive effect.40 These results are not sufficient for determining the optimum temperature because both l and the echo intensity, V, depend on the EPR lineshape, which is temperature dependent, as discussed in Section 3. Next, we discuss these two parameters.


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Fig. 9 W-band relaxation data on 0.05 mM Gd3+–DOTA in D2O/glycerold8 (7 : 3, v/v). (a) Temperature dependence of TM for a magnetic field set to the maximum of the central transition and 280 MHz higher. (b) Temperature dependence of the slow time constant, T1s, derived from saturation recovery data recorded at the maximum of the central transition. (c) The temperature dependence of exp(2tmax/TM)(T1s)1/2 for tmax = 5 ms for the two magnetic field set-ups given in (a).

5.1

The modulation depth

In the discussion of the modulation depth we assumed that both the observer and the pump pulses are on resonance with only one EPR transition per spin, (MS(A), MS(B)) - (MS(A) 1, MS(B)) and (MS0 (A), MS0 (B)) - (MS0 (A), MS0 (B) 1), respectively.47 This is a reasonable assumption considering the typical width of the sub-spectra of all transitions, except the central one. When D is large enough for most pairs the central transitions of spins A and B are well separated as well. Thus, the pump and observer pulses are restricted to spins B and A, respectively, and do not affect any transition of the partner spins. In addition, we assume that the pump pulse bandwidth is larger than odd. However, when D is small any pulse set to the central transition can affect both the A and B spins. Because the Gd3+ spectrum is a superposition of several transitions, the relative intensities of which are temperature, T, and spectrometer frequency, n0 (o0/2p), dependent, the observed l value is a weighted average of individual li values, each corresponding to a different transition with its own o1i and lineshape g(Do)i. Accordingly, the general expression for l becomes2,47 lðT; o0 ; DÞ ¼

2S X

li ðT; o0 ; DÞ ¼

2S X

i¼1

2S X

ðaðMS ÞpðMS Þ

i¼1

þ aðMS0 ÞpðMS0 ÞÞ

¼

DMS = 1 due to the pump pulse action, p(MS) and p(MS0 ) have to be scaled by the probability for DMS = 1 or DMS = 1 as given by the a(MS) and a(MS0 ) coefficients, respectively. For example, when pumping the |7/2i - |5/2i transition, p(5/2) will be scaled by a(5/2) = l0 (7/2 - 5/2)/[l0 (7/2 - 5/2) + l0 (5/2 - 3/2)]. l0 is defined in eqn (10). In this case a(7/2) = 1. Eqn (10) shows that the calculation of the modulation depth from the full EPR lineshape is complicated because it requires the spectrum to be deconvoluted into its individual transitions.47 To obtain some insight into the individual li values and their temperature and frequency dependence, we adapted a simplified approach that enables fast estimation of the spectrum lineshape without solving the full Hamiltonian problem. Briefly, we approximated the individual gi(Do) lineshapes as Gaussians centered at Do = 0 with a distribution of ci = 3nDG/4 (full width at half height), where n = 6, 4 and 2 for the (7/2 2 5/2), (5/2 2 3/2), (3/2 2 1/2) transitions, respectively. The width of the central transition was approximated by (2.75/16)[4(S(S + 1)) 3]DG2/n0 + d.50 Here DG is some effective ZFS parameter (DG B D) and d is the intrinsic linewidth, without the second-order ZFS broadening. This approximation is justified because of the featureless character of the Gd3+ spectrum due to the large distribution in D and E.52 Using this description of the EPR spectrum, we calculated the various li’s and the total l for typical W-band experimental conditions for Gd3+ tags with DG = 1000 MHz, a frequency separation of Dn = 90 MHz, and a pump pulse of 15 ns. These conditions are easily achieved on Q- and W-band spectrometers. In this particular case the central transition linewidth at W-band is narrow and it is not possible to place both the pump and observer pulses on the central transition. This is a typical situation for DOTA tags. Therefore, a choice has to be made: should the observer or pump pulse be set to the central transition? Fig. 10a (middle) shows the temperature dependence of l and the individual li values for npump set to the maximum of the central transition, whereas Fig. 10b (middle) shows the results for npump set at Dn = 90 MHz higher. This shows that setting npump to the central

ð

o1;i2 2 Oi t gi ðDoÞdðDoÞ sin p Oi2 2

ðaðMS ÞpðMS Þ þ aðMS0 ÞpðMS0 ÞÞli0

i¼1

(10) where p(MS) and p(MS0 ) are the populations of the levels corresponding to the i allowed MS - MS0 transition, affected by the pump pulse. As all spin states (except 7/2 and 7/2) can change by


Fig. 10 The individual Vi (top), li (center) and the product liVi (bottom) along with their sum, yielding l, V and lV calculated according to eqn (10) and (11) for 94.9 GHz, DG = 1000 MHz, tp = 15 ns and t0 = 15, 30, 30 ns for the following cases: (a) pump pulses on the central transition and observer pulses 90 MHz higher. (b) Observer pulses on the central transition and pump pulses 90 MHz higher. Color codes: |1/2i - |1/2i – blue, |3/2i |1/2i and |1/2i - |3/2i – light blue, |5/2i - |3/2i and |3/2i - |5/2i – red, |7/2i - |5/2i and |5/2i - |7/2i – green, sum – purple.


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transition yields the largest l value and that it is dominated by the contribution of the central transition down to 3 K. In this case, the contributions of the other transitions are small and thus they can be neglected. Under these conditions at 10 K, l(T,o0,D) B lcentral (T,o0,D) B 12%. As expected, a much lower l value (B4%) is obtained when the pump pulse is set outside the central transition. The width of the central transition is proportional to D2/n0 and therefore lcentral p [D2/n0]1. The temperature dependence enters through the populations of the MS = 1/2 levels. Accordingly, when setting the pump pulse to the central transition, l should increase with spectrometer frequency and decrease with D. l also increases with temperature until it reaches its maximal value of B0.2. Just on the basis of l, one would choose a temperature of 25 K, but then one has to worry about the effects of random flips caused by T1 or spectral diffusion.40,47 The calculated l values are significantly higher than those observed experimentally. One possible reason could be random spin flips caused by short T1 values, as discussed earlier,47 or other spectral diffusion processes. The probability of random flips owing to T1 is (1 exp(tmax/T1)), and it was shown that once T1 > 10tmax, the DEER trace is unaffected by such processes.47 In many relaxation measurements that we carried out at W-band, using saturation recovery, we found a biexponential recovery rate with a long time constant assigned to T1 and a shorter one, which at 10 K was about 10–50 ms. For a fast decay constant of 10 ms and tmax = 3 ms a 25% reduction in l is expected. Experimental evidence supporting this was observed for a peptide, melittin, labeled with two Gd3+ ions, where l was found to decrease as tmax and the temperature increase.40 From these data, we estimated, using the above relation, the time constant for the random flip to be B9 ms at 10 K and 4 ms at 25 K. The fast decay rates obtained from the saturation recovery measurements were 22 and 12 ms, respectively. These are a factor of 2–3 longer than estimated from the l reduction. We therefore suspect that there is another mechanism that contributes to the reduction in l; this will be discussed further in Section 6.2. 5.2

The echo intensity

V also depends on the EPR lineshape, which is temperature and frequency dependent. For S = 7/2, V receives contributions from all transitions and for the refocused echo used in the four-pulse DEER sequence, it can be described by eqn (11):47 V ðT;o0 ;DÞ /

2S X i¼1

Vi /

2S X

sin o1;i t0;1 sin2 o1;i t0;2 sin2 o1;i t0;3

In eqn (12) t0,1–3 correspond to the three observer pulses, respectively. Eqn (11) does not include echo reduction because of the pump pulse being on resonance with the central transition of some B spins,45 instantaneous diffusion, and the DEER background decay from the interpair dipolar interaction. The latter two should be small at the concentration normally used (0.05–0.1 mM). Neglecting the effects of T1 and TM, we calculated, using eqn (11), the individual Vi’s and their sum, V, as a function of T for the same DG values used for the evaluation of l and p/2 and p observer pulses of 15 and 30 ns, respectively. The results are shown in Fig. 10a (top) for the observer pulses set off the central transition, where the lower the temperature (down to 10 K), the more intense is the echo, as expected. For the other case, where the observer frequency is set to the central transition, shown in Fig. 10b (top), the contribution of the central transition to the echo dominates and a maximum echo is reached at B10 K. Fig. 10a (bottom) and b (bottom) present the product, lV, for the two cases discussed above, showing that the optimal temperature range is 8–12 K, which approaches the range suggested by T1 and TM for high tmax values (see Fig. 9). In addition, the calculations show that setting the observer pulse to the central transition frequency will yield a factor of B2 better DEER sensitivity, not considering instrumental limitations.47 Further SNR improvement is expected for this setting due to the higher TM of the central transition (see Fig. 9). Though such an example has been reported,60 so far, most reported DEER measurements were carried out with the pump pulse set to the maximum intensity of the central transition. The much lower l value obtained when observing the central transition requires very high stability of the spectrometer and a high dynamic range because a very small change is observed for a very large signal. For Q-band, and the same pulse durations and DG value, the lV values are similar to those at W-band but the optimum temperature is 4–5 K (see Fig. S2, ESI†). The above evaluation of lV should be considered as qualitative only because of the assumptions made in calculating V and l. The above considerations apply mostly for the weak coupling case, assuming that the high spin system can be treated as a superposition of effective single-spin systems with the pulses affecting only individual transitions. Complete density matrix calculations, taking into account the finite duration of the pulses and the complete spin Hamiltonian (eqn (5)), should be carried out for accurate evaluation of the modulation depth. Next, we discuss the potential effects of the ZFS.

i¼1 0

ðpðMS ÞpðMS ÞÞ;

(11)

where sin o1;i t0;1 sin2 o1;i t0;2 sin2 o1;i t0;3 2 ð 2

o1;i2 2 Oi o1;i o1;i 2 Oi t0;2 tp0;3 gi ðDoÞdðDoÞ sin Oi t0;1 sin sin ¼ Oi2 Oi2 2 Oi2 2 (12)


6. The effect of the ZFS Two aspects of the ZFS that influence the dipolar splitting should be considered. The first is the tilting of the quantization axis away from the direction of the external magnetic field. This has already been addressed in the context of ENDOR61 (electron-nuclear double resonance) and ESEEM (electron spin echo envelope modulation),62 where the nuclear frequencies are of interest. In these studies, the ZFS effect has been taken


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into account by introducing effective projection operators using 4hD perturbation theory for a small D, 1. It was concluded gbe B0 that for D r 1200 MHz, which applies to most Gd3+ complexes,52 the apparent DEER modulation frequency can deviate from odd by no more than 3–5% if the measurements are performed at Ka-band (B30 GHz) or higher.35 This was confirmed by the experimental results reported so far.35,39,42–44 The second effect arises from the pseudo-secular term of the dipolar interaction, which is neglected for the weak coupling regime (odd/2p o |nA nB|),39 as is usually encountered for a pair of nitroxides. Whereas the first type of distortions is effective for large D values, the second one is important for small D values. These two effects are discussed next. 6.1

The effect of the ZFS on the spin quantization axis

A second order perturbation derivation, based on the Hamiltonian in eqn (5), which focuses on how the ZFS affects the dipolar splitting under conditions of weak coupling, has been reported, and the general expression for dipolar splitting is given by39

i 1h ðBÞ ðAÞ ðBÞ ðAÞ odd ¼ Edd minitial ; minitial Edd minitial ; mfinal h (13) h

i ðBÞ

ðAÞ

ðBÞ

ðAÞ

Edd mfinal ; minitial Edd mfinal ; mfinal

where Edd corresponds to energy shifts owing to the dipolar interaction. The superscripts (A) and (B) correspond to each of the spins in the pair. The first term in brackets corresponds to the dipolar energy contribution to a particular transition where mA,B initial and mA,B final are the MS values of that particular EPR transition, where spin (A) changes its MS value and that of spin (B) remains constant. The second term corresponds to the same transition of spin (A) but the MS value of the second spin has changed owing to the application of the pump pulse. The expression for Edd(mAi ,mBj )/ h, given in the ESI† of ref. 39, is quite lengthy. For D = 0 for any transition pair, it is given by eqn (1). Representative simulations of the powder pattern of odd for a pair of Gd3+ ions (based on eqn 13) are shown in Fig. 11. For such a calculation the relevant parameters, in addition to the distance, are the ZFS interaction parameters, D and E, considered to be the same for both ions, the Euler angles (a,b,g) relating to the ZFS principal axis systems of the two ions, and the angles (yT,fT) representing the orientation of the inter-ion vector r with respect to the ZFS principal axis system of one of the ions. For simplicity we took an axially symmetric ZFS tensor, i.e. E = 0. In this case only the angle b should be considered and a = g = 0. To account for the distribution in the orientation of the principal axis system of the ZFS with respect to the molecular frame, usually encountered in Gd3+,52 we averaged b, yT, and fT over the range of 0 to 1801 at steps of 251. Because the calculations are rather lengthy, we did not average over D. The effect is well illustrated by a single D value. In the simulations we considered a situation where the pump pulse is set to the |1/2i - |1/2i transition, whereas the observer pulses are set to either |7/2i - |5/2i, |5/2i |3/2i, |3/2i - |1/2i, or |1/2i - |1/2i transitions. This is


Fig. 11 The effect of the ZFS on the dipolar powder pattern for the weak coupling case calculated using eqn (13) at W-band. In these calculations r = 2.5 nm, and the D value of the two spins was taken as equal. The angle between ZFS Z principal axes, b, and the orientation of the principal axis system of the dipolar axes with respect to the ZFS principal axis system, given by yT and fT, were averaged in steps of 251. This was done to account for the distribution of the principal axis system of the ZFS with respect to the molecular frame. In all calculations the pump pulse was set to the |1/2i |1/2i transitions and the observer pulses were set on any of the other transitions, as noted in the figure. In (a,c) D = 1500 MHz and in (b,d) D = 6000 MHz. The top panels (a,b) give the dipolar powder patterns, whereas the bottom ones (c,d) present the difference between the obtained powder pattern and that observed for D = 0. In each panel the individual spectra were shifted relative to each other by 0.2 (a,b) or 0.1 (c,d).

a representative of the experimental conditions. Other combinations involving MS > 0 values are not relevant because their populations at W-band are low. Fig. 11a shows the odd powder pattern obtained for a spectrometer frequency of 95 GHz, a distance of 2.5 nm, and D = 1500 MHz. Such a D value is at the high range for Gd3+ chelates.52 To highlight the effect of the ZFS on the dipolar frequencies, we show in Fig. 11c the difference between the obtained powder patterns and that corresponding to D = 0. As shown, the differences increase with the |MS| value, namely, the least effect is obtained when the observer pulse is set to |1/2i - |1/2i transition, whereas the largest deviation occurs for |7/2i - |5/2i. The latter, however, does not exceed 10%. The same powder patterns were obtained when the observer pulse was set to the central transition value and the pump pulse was set to all transitions mentioned above. Considering the much wider breadth of the |7/2i - |5/2i transition, its relative contribution to the A or B spins at 10 K is small, and therefore in practice the deviations do not exceed 2–3%. These results also show that setting both the observer and pump pulses to the central transition, if it is wide enough, will generate the least distortions. Fig. 11b shows the powder patterns for D = 6000 MHz; the corresponding differences from the D = 0 case are shown in Fig. 11d. Whereas the deviations for the observer pulse set to the central transition are negligible, for all other transitions


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they are significant and reach up to 40% for |7/2i - |5/2i. This shows that when lower spectrometer frequencies such as Ka-band are used, applying the pump and observer pulses to the central transition is preferred. Since the central line will be broader by a factor of B3 compared to W-band, this will be easier to achieve. 6.2

Deviation from the weak coupling condition

In Section 6.1 it was assumed that there is no overlap between the excitation profiles of spins A and B. This usually holds for S = 1/2 nitroxide spins at X-band because the spectral width allows sufficiently large Dn values. However, the case of an S = 7/2 spin pair is more complicated. Let us consider the most common way of setting up the pulses, with the pump pulse on resonance with the maximum of the |1/2 - |1/2i transition of the B spins and the observer pulse set to 70–100 MHz higher or lower frequencies. At this position the major contribution to the A spin echo will come from the |3/2 - |1/2i, |5/2 |3/2i and |7/2 - |5/2i transitions, the first one having the largest contribution (see Fig. 2b and 10). Owing to the broad powder pattern of these transitions with respect to the pulse bandwidth, the chance of B spins contributing to the echo at the observer frequency is slim. Similarly, when D is large, the width of the sub-spectrum of the central transition increases such that the probability that the pump pulse also affects A spins is low. In contrast, when D is relatively small, as desired for high EPR sensitivity, the sub-spectrum of central transition is narrow and there is a good chance that the pump pulse will also affect the |1/2i 2 |1/2i transitions of some A spins. Since this transition has a common energy level with the observed |3/2 - |1/2i transition of A spins, this will cause some reduction in the |3/2 - |1/2i contributions to the echo because part of the coherence is transferred to other elements of the density matrix. Accordingly, some instantaneous decrease in the echo intensity is expected just by applying the pump pulse.45 Experimentally, the echo reduction we observed at W-band is usually around 20% and this affects the sensitivity of the experiment. Similarly, also in the case where the observer pulse is set to the central transition, it will generate coherences corresponding to the central transition of spin B and may reduce the net effect of the pump pulse, given by l. This problem is less severe when the ZFS is large because the central transition cannot be fully excited, but in such cases the sensitivity is reduced due to the broader EPR spectrum. A more severe consequence of a small D value is that the weak coupling approximation does not hold any longer. This is due to the weak dependence of the |1/2i - |1/2i transitions D2 on D, / . As a result for many orientations the condition n 0 odd oA 1 1 oB 1 1 is not fulfilled. This will result in a 2!2

2!2

shifting of energy levels MS = 1/2 of both spins and as a consequence the splitting between consecutive lines within the octet signal of the observed |3/2 - |1/2i transition of A spin is no longer odd for all lines. This variation in the splitting will


be manifested in frequencies in the DEER that deviate from odd, as given by eqn (1) and data analysis using DeerAnalysis, which uses the kernel given in eqn (2), will lead to artificially broadened distance distributions. Under these conditions, transition probabilities change as well and this will consequently affect the modulation depth, l. To account for this variation the pseudo-secular term of the dipolar interaction that was neglected in the calculation of the Edd(mAi ,mBj ) values in eqn (13) should be taken into account as suggested earlier.39 Detailed simulations are currently being carried out by our group in order to evaluate this effect more quantitatively and to search for the range of distances and D values that will tolerate this effect. When the distance distribution is large, as is often observed in proteins, this effect is expected to be hidden within the distance distribution, as shown by the experimental data presented in Section 4.

7. Gd3+–nitroxide distance measurements Whereas Gd3+ is a very attractive observer spin in a DEER experiment at high fields owing to its intense central transition, of its short T1 and its reasonably long TM, it is less attractive as a pumped spin. Although it allows for short MW pulses, its modulation depth is rather low. One way to increase l is to replace one of the Gd3+ labels in the Gd3+–Gd3+ pair with a nitroxide and set the pump pulse on the nitroxide. Here no change in V is expected, but the narrower spectral width of the nitroxide spectrum, compared with that of Gd3+, will lead to an increase in l, provided that short enough pulses can be applied. Since the repetition rate of the DEER experiment depends on the T1 value of the observed spins, the long T1 value of the nitroxide will not be an obstacle. DEER distance measurements on a Gd3+–nitroxide rigid model were first reported at X- and Q-bands.63 The SNR at X-band was considerably worse than at Q-band, as expected considering the spectrometer frequency dependence of the central transition. In addition, at X-band the ZFS contributions should be substantial. Clear modulations were observed and data analysis yielded the expected distance, 2.45 nm. Under the best Q-band experimental conditions, a pump pulse of 12 ns generated l = 0.3. In this work it was also demonstrated that nitroxide–nitroxide distance measurements on the same sample can be used to probe the formation of dimers when the concentration is high. When Gd3+–nitroxide DEER is carried out at Q-band frequencies and higher, the frequency separation between the maximum of the nitroxide spectrum and the central transition of Gd3+ is sufficient to guarantee weak coupling, and one only has to worry about the ZFS tilting the quantization axis. Detailed calculations showed that for typical Gd3+ D values the broadening introduced in distance distributions obtained using DeerAnalysis is within the experimental width expected in biomolecules.45 Gd3+–nitroxide distance measurements were also reported for proteins; the first was at W-band on a homodimer of the S114C/C157S double mutant of the ERp29 chaperone, where


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protein molecules labeled with a C1–Gd3+ tag and a nitroxide label were mixed.60 The final sample consisted of a mixture of ERp29 dimers with the following label composition: two Gd3+ tags (25 mM, 25%), two nitroxide labels (25 mM, 25%), and one nitroxide and one Gd3+ tag (50 mM, 50%). By choosing the appropriate experimental conditions, namely, temperature, pulse durations, and repetition rates, it was possible to selectively determine Gd3+–nitroxide, nitroxide–nitroxide, and Gd3+–Gd3+ distance distributions. At W-band the frequency separation between the maxima of the Gd3+ and the nitroxide spectra is very large, B700 MHz, compared with 300 MHz at Q-band.64 Thus, optimal sensitivity cannot be achieved with W-band spectrometers that use cylindrical cavities with a limited bandwidth because this forces setting the observer pulse off the Gd3+ central transition, thus considerably compromising sensitivity.60 Another difficulty in using this type of measurements is that applying the pump pulse for standard Dn = values of 65–100 MHz leads to a substantial echo intensity, V, reduction. This is due to the very strong direct off-resonance effect of the pump pulse on the observer spins that is very pronounced due to the high transition probability of the high-spin Gd3+ ion.60 A pump pulse with a flip angle of p for the nitroxide spins results in an B4p pulse for the Gd3+ spins. This difference produced a much stronger echo reduction compared to DEER applied to two Gd3+ labels, where the echo reduction effect about 20%. Another reason for this echo reduction effect is that the pump pulse also excites some Gd3+ transitions that share a level with the Gd3+ observer spins,45 as mentioned earlier. Hence, to obtain DEER data with an acceptable S/N ratio, the duration of the pump pulse was reduced.60,64 This problem was solved by using a dual-mode cavity that matches the B700 MHz frequency separation between the Gd3+ and nitroxide maxima.65,66 This dramatically increased the sensitivity of the DEER measurements, allowing evolution times as long as 12 ms in a protein,66 as shown in Fig. 12. This long dipolar evolution time indicates that distances greater than 8 nm can be accessed. In addition, orientation selection could be resolved and analyzed, thus providing additional structural information. In the case of W-band DEER on a Gd3+–nitroxide pair, only two angles and their distributions have to be determined, which is a much simpler problem to solve than the five angles and their distributions associated with two nitroxide labels.25

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Gd3+–nitroxide, Gd3+–Gd3+, and nitroxide–nitroxide Q-band DEER distance measurements were demonstrated on nanoparticles functionalized with nitroxide radicals and Gd3+–DTPA chelates.45 Gd3+–nitroxide distance measurements were also reported for a WALP peptide forming a trans-membrane helix in model membranes.64 Here a Gd3+ chelate was attached to one end of the peptide through an additional modified lysine residue. The nitroxide spin label was attached at different positions along the helix via cysteine mutations and the distances obtained agreed well with those expected for an a-helix. In addition, nitroxide–nitroxide distance measurements carried out on the same samples were used to probe oligomerization. Finally, labeling a single protein molecule with two types of labels is synthetically demanding and to obtain 100% selectivity one has to use orthogonal labeling schemes. Recently it has been demonstrated that this is possible in a protein.67 In all these cases no orientation selection was reported. Although orthogonal spin labeling may be too demanding and therefore less routine, it is possible to envision much simpler applications such as distance measurements aimed at determining the interaction between different biomolecules. In this case one molecule can be labeled with Gd3+ and the other with a nitroxide. Gd3+ labels that are bulkier than nitroxides can be used to label surface sites in one protein, whereas inner sites in the other protein could be labeled with a nitroxide. In principle, one could also envision measurements between a doubly labeled protein with either two nitroxides or two Gd3+ tags and a second protein or DNA labeled with a nitroxide or a Gd3+, respectively. Then, by conducting selective Gd3+–Gd3+ and Gd3+–nitroxide and nitroxide–nitroxide distance measurements, one could obtain two intermolecular distances as well as one intramolecular distance from one sample.

8. Future outlook In this Perspective, the prospects of new spin-labeling schemes based on half-integer high-spin metal ions, particularly Gd3+, for nanoscale distance measurements have been reviewed. The Gd3+ spectral properties at spectrometer frequencies higher than ca. 30 GHz make Gd3+ tags an attractive general purpose spin label for high sensitivity distance measurements in biomolecules. To date, DEER measurements have been carried out

Fig. 12 W-band DEER measurements on mixed Gd3+–nitroxide labeled ERp29 dimers at 10 K. (a) Time domain traces after background removal. (b) Their corresponding Fourier transforms (solid lines). Best-fit simulations are shown as dashed lines. (c) Distance distribution obtained with Deer Analysis. Reprinted ref. 66, Copyright 1990, with permission from Elsevier.



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at Ka-, Q-, and W-band frequencies, showing that distances of up to 6 nm can be accessed routinely in frozen aqueous solution and the results indicate that this can be extended to 8 nm. Moreover, measurements in membranes are feasible as well. This has important implications in terms of future functional studies of membrane proteins. It has also been established that the data analysis can be carried out with the existing DeerAnalysis software and that distance distributions can be readily obtained, for systems with finite flexibility and D values smaller than B400 mT. The absolute sensitivity of Gd3–Gd3+ DEER measurements is high, particularly at W-band, where the sample amount required is >0.15 nmol. For Gd3+–nitroxide measurements the absolute sensitivity may be even higher, provided that the right cavity is used at W-band. Using high power, broadband W-band spectrometers22 featuring high concentration sensitivity, concentrations as low as 1 mM, though with higher volumes, may be accessed. A further improvement in sensitivity is expected by applying shaped pulses and novel sequences.18,19 Considering the narrow central transition, applying the DQC method68 for measuring distances seems highly attractive and may result in better SNR than DEER. Here the challenge will be to separate inter- and intra-Gd3+ doublequantum coherences. The low modulation depth observed for Gd3+–Gd3+ pairs is advantageous when oligomers are concerned, since there are significant dipolar interactions between more than two spins. In such cases, when the modulation depth is substantial the distance distribution derived from DeerAnalysis contains artifacts, because of the so-called multi-spin effects, which are proportional to l2, l3, etc.69 For nitroxides this effect has been shown to be significant70 and one way to remove the artifacts is to reduce l. This, however, will reduce sensitivity. For Gd3+– Gd3+ DEER, where the major contribution to the sensitivity comes from V, l is very small and therefore multi-spin artifacts are expected to be minimal. Although there has been remarkable progress in this approach since the first Gd3+–Gd3+ DEER report, more work is still needed to fully realize this approach and recognize its limitations. In the context of Gd3–Gd3+ DEER, the effects of the pseudo-secular terms of the dipolar interaction for small D values have to be quantitatively evaluated and guidelines for the optimal range of D values for Q- and W-band measurements should be given. For small D values, as encountered for DOTA tags, and short distances (B2–3 nm), where the pseudo-secular terms of the dipolar interaction become significant, Q-band Gd3+–Gd3+ DEER measurements seem to be preferable due to the broader central transition. Similarly, relaxation studies should be carried out to better understand the dominating phase relaxation mechanisms so that a tag with a long TM can be designed. So far, because of their relatively large size (except for DPA derivatives) attachment of Gd3+ tags has been limited to the external surface of the protein; it is still not clear whether such tags can be used to label internal sites in the proteins. In terms of tag development, efforts should be joined with those involved in tag development for paramagnetic NMR applications, thus seeking for multifunctional tags for both NMR and


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EPR applications.43 Finally, the direction of high-field (>W-band) CW EPR for distance measurements at temperatures approaching room temperature is currently unexplored and seems promising. The combined efforts in instrumental and methodology development for improved sensitivity, along with tag design, will hopefully lead to the use of this new labeling approach for in-cell structural studies owing to the higher stability of Gd3+ in the cell compared with nitroxides.

Acknowledgements I thank Arnold Raitsimring and Gottfried Otting for their continuous, wonderful collaboration in the Gd3+ spin-labeling endeavor and for their inspiration. I thank Akiva Feintuch for his numerous contributions to both spectrometer development and maintenance and for fundamental understanding regarding the spin physics of Gd3+ DEER. I also wish to thank my former and current group members who carried out the work presented here: Alexey Potapov, Michal Gordon-Grossman, Ilia Kaminker, Debamalya Banerjee, Erez Matalon and Arina Dalaloyan. I am indebted to David Milstein, Chidambaram Gunanathan, Hirosama Yagi, Tom Meade, and Ying Song for synthesizing the bis-Gd3+ models, the labeled proteins, and DNA. I greatly appreciate the efforts of Yaacov Lipkin, Koby Zibzener, David Leibovitch, and Yehoshua Gorodetski in building our high power W-band microwave bridge, and the efforts of Boris Epel for his constant support of the software controlling the spectrometer. This research was supported by the Binational USAIsrael Science Foundation, and the Israel Science Foundation (ISF), and was made in part possible by the historic generosity of the Harold Perlman Family. D.G. holds the Erich Klieger Professorial Chair in Chemical Physics.

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Pulsed EPR distance measurements in soluble proteins by site-directed spin labeling (SDSL).

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PELDOR.

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