Spectroscopically orthogonal spin labels and distance measurements in biomolecules

Maxim Yulikov DOI: 10.1039/9781782620280-00001

Essential details of two techniques for distance measurements between non-identical spin labels are summarized. One technique is based on double electron–electron resonance (DEER) between Gd(III) ions and nitroxide radicals. The other technique is based on indirect measurements of stochastic dipolar interaction between Ln(III) ions and organic radicals via the change of longitudinal relaxation of the latter species. Combination of these techniques with double electron–electron resonance in pairs of identical spin labels (nitroxide–nitroxide or Gd(III)–Gd(III)) allows to suggest a new experimental strategy for multiple distance measurements in orthogonally-labelled samples. General discussion of advantages and disadvantages of the new strategy for studies of biomacromolecules and their complexes is given along with illustrative experimental examples. In particular, performance of Gd(III)–nitroxide DEER is compared to other possible combinations of nonidentical spin label pairs, while relaxation enhancement in pairs Fe(III)–organic radical is compared to the case of Dy(III)–nitroxide pairs.

1 Introduction

EPR-based techniques to measure nanometre range distances are nowadays recognized as a valuable tool in studies of structure and conformational changes of biomacromolecules. A typical approach uses some type of nitroxide-based spin labels, which are selectively attached to specific sites. The distances between pairs of these spin labels are then measured by some appropriate pulse EPR technique, most commonly, by the dead-time-free 4-pulse double electron –electron resonance (DEER) experiment.

As the field of structural and molecular biology proceeds towards more and more demanding objects, such as multi-subunit complexes in solution or in lipid membranes, development of appropriate EPR methodologies becomes necessary. For instance, while being proved to be robust and sensitive, the nitroxide-based approach does not allow distinguishing between specific labelling sites, because two nitroxide labels are typically very difficult to distinguish spectroscopically. Thus, measurement of the properties of the local environment for a spin label at a particular site requires additional singly-labelled samples. Furthermore, in multiple-subunit systems one is restricted to labelling only two sites per biomolecular complex, otherwise interpretation of the distance distribution gets difficult, as all pairwise distance distributions overlap and cannot be separated from each other. Further difficulties may be caused by the appearance of combination frequencies in the DEER experiment on multiply-labelled biomolecules.

The number of labelled sites and, thus, the accessible number of distances can be increased by employing non-identical types of spin labels. In this chapter we will discuss an approach of using distinct types of labels for each labelling site in a biomolecule or biomolecular complex under study. We will mainly concentrate on combining nitroxide radicals with chelate complexes of different lanthanide ions, but other spectroscopic selection options tested so far will be shortly reviewed as well. After providing general introduction in the following part of this section, Sections 2 and 3 will give details of two particular distance measurement approaches for the lanthanide–nitroxide pairs. In Section 4 we will attempt to formulate perspectives of this EPR methodology and discuss its current state of development.

1.1 Brief overview of the spectroscopically-selective spin label pairs for DEER spectroscopy

The idea of having two non-identical spin labels that can be distinguished spectroscopically appeared rather early after DEER (sometimes also called PELDOR) and related techniques have attracted considerable attention. It has been demonstrated that 14N/15N labelling of the nitroxide radicals can provide a difference in the nitroxide EPR spectra that is sufficient to distinguish between the two types of labels. In another publication, a selective detection of Cu(II)–nitroxide and nitroxide–nitroxide distance was reported. Later, when Gd(III) chelate complexes were proposed as possible paramagnetic labels for the DEER-based distance measurements, the performance of the DEER experiment was also studied in Gd(III)–nitroxide pairs.

The use of Gd(III) chelate complexes as the second spin label instead of 15N-labelled nitroxides or Cu(II) complexes offers important advantages. Spectroscopic separation of the signals from nitroxide radicals and Gd(III) centres relies to a lesser extent on the difference in resonance fields for the two types of paramagnetic species. The main factors that allow spectroscopic separation are the 2–3 orders of magnitude difference in longitudinal relaxation time (at optimum repetition rate for Gd(III) species nitroxide radicals are nearly completely saturated) and the difference in the transition moments between 'high-spin' (S = 7/2) Gd(III) centres and 'low-spin' (S = 1/2) nitroxide radicals.

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For the central, most narrow | 1/2 > [left right arrow] | + 1/2> transition of Gd(III) the ratio of the corresponding transition moment to the one of a nitroxide radical is exactly four, which perfectly suppresses the Gd(III) signal for the optimal pulse settings of the nitroxide radicals: the Hahn echo detection sequence (π/2–π-echo) tuned for nitroxide radicals would correspond to a 2π–4π-echo sequence on the central Gd(III) transition, which would ideally produce a zero echo amplitude. For other single-quantum transitions of Gd(III) the corresponding transition moments of the spin operators are somewhat smaller than the one for the central transition. Still, the overall experimentally observed selection is nearly perfect. In contrast to this, spectroscopic selection in 14N–15N nitroxide pairs nearly exclusively depends on the (only partial) spectral separation of the signals and is not quantitatively perfect. In Cu(II)–nitroxide pairs the relaxation time difference can be used as well, but Cu(II) is an S = 1/2 system, and thus the transition-moment-based selection is not available in this case. The Cu(III) EPR spectrum at Q-band and higher detection frequencies is also broader than that of the | — 1/2 > [left right arrow] | + 1/2 > transition of Gd(III) centres. Therefore, more Gd(III) species can be excited by a microwave pulse of a given bandwidth, thus providing a sensitivity advantage. Cu(II) centres also typically have a strong spectroscopic separation of different orientations of the complex with respect to the applied static magnetic field. In contrast, Gd(III) chelates seem to have no orientation selectivity at any spectral position. This significantly simplifies applications where distance information is required. The orientation information can still be assessed, if necessary, via selective excitation of nitroxide labels.

A further type of spin labels that can be sufficiently well separated spectroscopically both from the nitroxide radicals and the Gd(III) centres are the trityl radicals that were recently reconsidered for pulse EPR distance measurements. In this case, the spectroscopic separation of the trityl and nitroxide radicals is mainly due to the difference in resonance fields. Still, due to the very narrow spectrum of trityl such a separation can be sufficiently good. A disadvantage of trityl radical is its large size that might limit the range of biochemical applications of such a spin label.

1.2 Extending the spectroscopically-selective spin labels approach to the relaxation-based distance measurements

Another line of spectroscopic selection options appears if the second spin label is essentially invisible in the pulse EPR measurements. This is the case if chelate complexes of any trivalent paramagnetic lanthanide ions [except Gd(III)] are used as spin labels. Those lanthanide ions relax fast enough to be non-detectable in any pulse EPR experiment at least down to ~10 K. Such a spin label would not lead to any spectral crowding problem, but its dipole–dipole interaction with nitroxide radicals can still be measured via the relaxation enhancement (RE) effect.

The relaxation enhancement-based distance measurements were initially proposed for continuous wave (CW) EPR. The CW EPR-based techniques can be utilized at ambient temperature, which is closer to the physiological conditions. (In contrast to this, pulse EPR experiments are almost exclusively done at low temperatures, with the solvent in a frozen glassy state.) Interestingly, applications of lanthanide ions as relaxing agents were suggested in the early research. Later, however, the CW EPR approaches based on Ni or Cr complexes became more popular. These techniques are mostly considered to be qualitative, in particular due to the interference between relaxation enhancement and molecular motion.

In a series of papers of S. S. Eaton, G. R. Eaton and co-workers, distances from organic radicals to a transition metal centre (mainly Fe(III)) were measured by pulse EPR relaxation techniques. These reports and contributions from other groups up to the year 2000 were reviewed, but to date did not lead to a broad range of applications, partially due to a quick spread of DEER-based approaches at about the same time. It is also important to mention that initially RE-based distance measurements were mainly concentrating on protein molecules, naturally containing transition metal centres (e.g., methemoglobin), while the DEER technique was from the beginning combined with site -directed spin labelling (SDSL) which allowed for studying a much broader range of systems. Still, several interesting papers on RE were since published by different groups, in particular, clarifying some important theoretical points, data analysis procedures, and testing SDSL approaches and new label types. While static dipolar interaction exploited in the DEER technique allows for a more straightforward distance calculation, the RE technique requires certain assumptions on the characteristic correlation time for stochastic fluctuations of the fast relaxing spin label. Moreover, the presence of further paramagnetic centres in the sample may cause interference with the RE effect in a given spin-label pair. Thus, further development of the RE -based distance measurement technique required calibration against well-established DEER measurements. In this case use of lanthanide tags provides a very convenient option of exchanging fast relaxing ions by slowly relaxing Gd(III) and thus calibrating RE distance measurements against DEER in geometrically nearly identical Gd(III)–nitroxide pairs. A detailed analysis of this technique and discussion of its precision and possible application area will be given in Section 3.

To summarize, combination of lanthanide chelate complexes and nitroxide radicals allows for a series of different types of distance measurements presented schematically in Fig. 1 (along with schematic representation of two possible experimental situations, where such a scheme would be useful). Inclusion of further labels, such as trityl, into this scheme would be rather straightforward. The scheme consists of distance measurements between identical spin labels (DEER in nitroxide–nitroxide and Gd(III)–Gd(III) spin pairs) and between nonidentical spin labels (RE experiment in the nitroxide–Dy(III) pairs or DEER in the nitroxide–Gd(III) pairs). The newest developments for the DEER technique on pairs of identical spin labels were recently reviewed. In the following two sections we will overview specific details of the distance measurements between non-identical labels. After that, in the final section, a general discussion of the current state and future perspectives of this new experimental strategy will be given.

2 DEER in Gd(III)–nitroxide pairs

2.1 Theoretical and practical aspects of the technique

The echo-detected EPR spectra simulated for nitroxide radicals and Gd(III)–DOTA complexes at the three most common detection bands (X, Q and W band, i.e., ~9.5, ~34 and ~95 GHz detection frequency) are presented in Fig. 2. The spectrum of nitroxide radicals mainly consists of three anisotropically broadened sub-spectra corresponding to the three spin states of a paramagnetic 14N nucleus (mI = +1, 0, -1). There is interplay between g- and hyperfine anisotropy for each of the three sub-spectra. As a result, at X band the central one (mI = 0) has least cumulative anisotropy and the maximum intensity in the nitroxide spectrum is close to its centre. At Q band the low-field sub-spectrum (mI = +1) gets least broadened as the g- and hyperfine anisotropies nearly compensate each other in that case. At W and higher bands the g-anisotropy dominates the spectrum, all three sub-spectra strongly overlap (except of the gz region) and the maximum of the spectrum is again in the centre close to the gy position.

For so called orientation selection measurements (see Section 2.2) it is important how well different orientations of g- and hyperfine tensors are separated in the EPR spectrum. While different hyperfine sub-spectra overlap stronger in the order X < Q < W band, the spectral positions of different principal components of g- and hyperfine tensors move closer to each other in this order, and the spectral resolution increases with the increase of the detection frequency. Therefore, among these three bands, despite stronger sub-spectra overlap, the measurements at W band offer best spectral selectivity for nitroxide orientations.