Side-directed spin labeling

First side-directed spin labeling (SDSL) studies date back to the early 1970s. There, tRNAs were subjected to nitroxide labeling via either naturally occurring rare nucleobases, e.g., 2-thio-uridine, enzymatically introduced nucleobases such as... 

Abstract EPR spectroscopy of site-directed spin labeled membrane proteins is at present a common and valuable biophysical tool to study structural details and conformational transitions under conditions relevant to function. EPR is considered a complementary approach to X-ray crystallography and NMR because it provides detailed information on (1) side chain dynamics with an exquisite sensitivity for flexible regions, (2) polarity and water accessibility profiles across the membrane bilayer, and (3) distances between two spin labeled side chains during protein functioning. Despite the drawback of requiring site-directed mutagenesis for each new piece of information to be collected, EPR can be applied to any complex membrane protein system, independently of its size. This chapter describes the state of the art in the application of site-directed spin labeling (SDSL) EPR to membrane proteins, with specific focus on the different types of information which can be obtained with continuous wave and pulsed techniques. 

An extension of site-directed spin labeling is to monitor H-ESEEM from a deuterated amino acid (valine or leucine) that is introduced at a fixed position, i, in the peptide sequence. The nitroxide spin label is then attached at a cysteine residue that is stepped sj tematically away from the deuterated residue. For an a-helical peptide, H-ESEEM is observed when the spin label is at position z- -3, i.e., in register with the deuterated residue, but not at position i + 2. For a (Tsheet peptide, on the other hand, H-ESEEM is observed when the spin label is at position i -I- 2 on the same side of the p-sheet as the deuterated residue, but not at position i -I- 3 on the opposite side of the p-sheet. Therefore the H-ESEEM amplitude, which is inversely proportional to the sixth power of the distance between electron and nuclear spins, directly reflects the characteristic periodicity of the peptide secondary structure, in the vicinity of the deuterated residue. 

Fig. 12. (A) Structural model of rhodopsin showing the location of cysteine substitution mutants in TM7 (306-309) and H8 (310-321). Ca-C bonds indicate the direction in which the side chain projects. The bonds are shaded according to local maxima (white), local minima (black), and intermediate (gray) in 11(02). Cysteine residues that were unreactive to the spin label reagent and 4-PDS are marked by black spheres at the corresponding Ca. (B) /SA for the A and B molecules (upper panel), n(C>2) and FI(NiEDDA) (center panel), and PDs (lower panel) for the sequence 306-321.

As for Cl, the global topology of the TM7-H8 sequence was mapped in detail by direct distance measurements between pairs of spin labels (A1 tenbach et al., 2001c) and by disulfide cross-linking (Klein-Seetharaman et al., 2001). For direct distance measurement using spin-spin interactions, a reference R1 side chain was placed at site 65 in Cl, and a second R1 placed at each site in the sequence 306-316. Figure 14 shows the pairs and indicates the interspin distances... 

Having an accurate distance determination between the NO groups of the spin labeled side chains stUl does not directly convey the structural information at the level of the backbone of the protein which would be required for modeling structures and complexes at high resolution. To correlate the spin-spin distance constraints to the backbone-backbone distances requires modeling. At the present time, modeling approaches combining sparse accurate distance constraints to macromolecular structures are under development [7, 75]. 

SDSL EPR is sensitive to flexible regions of proteins and to dynamical changes, and can be used to measure water accessibility profiles and accurate distances between spin labeled side chains. The wealth of information that can thus be obtained makes SDSL EPR a direct tool to access conformational changes of proteins. The bridge... 

In direct detection experiments, several approaches were used to achieve virtual decoupling (to remove homonuclear one-bond carbon-carbon couplings) such as IPAP schemes (in-phase anti-phase) [150-152], in which two FIDs for each increment are recorded and stored separately, one for in-phase and another for antiphase, the two components being combined to remove the splitting. An alternative is S E schemes [152, 153] (spin-state selective excitation), in which two different experiments are performed with one being absorptive and another dispersive. One or more of these building blocks (IPAP and S E) can be implemented in any experiments based on direct detection. A set of based experiments, which can be used for the assignment of backbone and side-chains of labeled... 

Fig. 10. Cartoons depicting situations in which a spin-labeled polymer may yield the types of local director distribution shapes shown in Fig. 9. (a) Director perpendicular to polymer chain direction, determined by interactions between spin-label and liquid-crystalline side chains. Different ordering around two axes leads to the elongated distributions shown at the top of Fig. 9b Director parallel to main-chain direction, with spin label held at a fixed angle with respect to the chain, leading to the conical distributions shown at the bottom of Fig. 9.

We now turn to the t g configuration but we already know what we expect to find—those terms which appear at the weak field side of Fig. 7.20 and which have not yet been obtained. That is, we are looking for a T g term, the only spin triplet unaccounted for. As Table 7.4 shows, the direct product Tig X Tig = Aig A Eg- - T g "H T23. We expect that the addition of spin labels will lead to Aig + Eg + Tig + T2g. Is this correct A partial check can be made by counting two-electron wavefunctions. The configuration t g implies (6 x 5) = 15 two-electron wavefunctions. This is the number implicit in Aig + Eg A Tig T2g (14-24-94-3 = 15). It is reasonable then, to add the label to the line of slope —f A at the strong field limit of Fig. 7.20. 

Spin a pencil on a table N times. Each time it stops, the pencil points in one of four possible directions toward the quadrant facing north (n), east (e), south (5), or west (w). Count the niunber of times that the pencil points in each direction label those numbers tin, tie, tis, and tiw Spinning a pencil and counting orientations is analogous to rolling a die with four sides labeled n, e, s or w. Each roll of that die determines the orientation of one pencil or... 

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