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....