Distance between spin labels, determination

Persson, M., Harbridge, J. R., Hammerstrom, P., Mitri, R., Ma rtenson, L-G., Carlson, U., Eaton, G., and Eaton, S. (2001) Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II, Biophys. J. 80, 2886-2897. 

Stainhoff, H.-J., Radzwill, N., Thevis, W., Lenz, V., Brandenburg, D., Antson, A., Dodson, G., and Wollmer, A. (1997) Determination of interspin distances between spin labels attached to insulin comparison of electron paramagnetic resonance data with the X-ray structure, Biophys. J. 73,3287-3298. 

Other ESR and NMR studies have also been dealt with in Section VI. Gallo et al. (1971) and Teichberg et al. (1974) used ESR to explore the binding of metals to lysozyme. In addition, Berliner et al. (1983) used NMR as well as ESR to study the Mn(II) binding site of a-lactalbumin. More recently, Musci et al. (1987) used ESR and NMR to determine certain intramolecular distances between spin-labeled Met-90 and the metal binding site, as well as certain resolvable protons. 

Sucdnimidyl-2,2,5,5-tetramethyl-3-pyrrolin-l-oxyl-3-carboxylate was reacted with lysozyme to give labeling of 2.3 or 1.8 mol/mol of protein. ESR sjrectra were measured at — 160°C. The parameter dild (Likhtenshtein, 1976), a ratio of amplitudes, describes the extent of spin-spin interaction. The estimate of average distance between spins was made from the value of d ld by use of the plot of Fig. 15 of Likhtenshtein (1976). The separation of the two outer hyperfine extrema Ai ) is determined by the mobility of the spin probe and its environment. (From unpublished data of P.-H. Yang, G. Tollin, and J. A. Rupley, with permission.)... 

The spectra were obtained at a frequency of 100 MHz and with a MAS frequency of 12.487 kHz corresponding to the n = 1 rotational resonance that is the spinning speed was equal to the difference in isotropic shifts between the labeled sites. The time shown denotes the interval between inversion of the labeled methylene resonance, which occurs immediately after cross polarization, and application of the observation pulse. Thus, 0 ms corresponds to the initial nonequilibrium state established by the selective inversion. The rate of this decrease may be modeled to determine the distance between the labeled sites (Fig. 23.24 (bottom)). Comparison of magnetization exchange data (filled circle) for a37-38 along with the calculated curves for four distances 4.74 A (dotted line), 4.0 A (dashed line), 3.9 A (solid line) and 3.8 A (dot-dashed line). The 4.74 A distance would be expected for an idealized antiparallel j8-strand. The best fit to the data is 3.9 A. This result is for the undiluted a37-38 sample. To eliminate possible effects due to intermolecular interactions, measurements were performed on isotopically pure samples and on samples in which the doubly labeled peptide was diluted 1 5 and 1 10 in unlabeled peptide. This produced a corrected distance for a37-38 of 4.0 A and in all cases, the distance could be defined to within... 

Having demonstrated the achievement of high-resolution sohd state NMR capability, the authors describe experiments that combine the high-resolution aspect of MAS NMR with methods that retain the structure and/or dynamic information inherent in the anisotropic interactions. Rotational-echo double resonance (REDOR) allows the determination of D between isolated heteronuclear spin pairs. D is related simply and without approximation to intemuclear separation. Hence, REDOR makes possible the unambiguous direct determination of intemuclear distance between the labeled spin pair, independent of pair orientation, i. e., in amorphous and /or microaystaUine solids, and extends our abihty to quantitatively explore complex materials. It is also possible to extract intemuclear distance from homonuclear dipolar coupled spin pairs, and these experiments are also reviewed. 

Radicals normally do not occur in peptides but they can be generated by irradiation at low temperature with UV, electrons or X-rays, usually at the a-carbon atom, and then can be recognized by ESR. There are some radicals that are stable at ambient temperature, for instance N-oxides with the structural element C-NO-C. In biochemistry, e.g. derivatives of 2,2,5,5-tetramethyl-pyrrolidine-l-oxide are being used as spin labels, conjugated to bioactive compounds. In this way ESR can localize the position of possible receptor sites. For instance, in hormone research the distances in the neurophysin complex between spin-labeled small peptides, models of oxytocin, have been determined [27]. 

Fourier Deconvolution. A synthetic 30-residue polypeptide was synthesized as an electron-transfer protein.42 A spin label was attached at position 21 and the distance between the two labels in a dimer was determined by Fourier deconvolution of the CW line-shape in frozen solution. Based on the known geometry of the label, it was calculated that the interspin distance of 22.5 A corresponded to 13.5 + 0.9 A between the Ca carbons on the two polypeptide chains. 

Amyloid fibrils formed from a-synuclein have been found in Lewy bodies of patients with Parkinson s disease.64 Spin labels were introduced at 36 positions between amino acid 5 and 136 of a-synuclein. Distributions of interspin distances between the same labels on neighboring chains were determined by analysis of CW line-shapes in solution. For the fibrils analysis of the percent of molecules with distances <15 A, 15-20 A, and > 20 A revealed a highly ordered and specifically folded core region of 70 amino acids ( residues 34 to 101). In contrast, the N terminus region is structurally heterogeneous and the C terminus appears to be completely unfolded. 

Thus, we have determined the distances between the adenylyl moiety and the two divalent metal ion binding sites on glutamine synthetase by 13C and 3 P NMR, spin-labeled EPR, and fluorescence energy transfer methods. The results obtained from each method are in good agreement. The data show that the adenylyl regulatory site is close to the catalytic site (12-20 A). Additional data on the rotational correlation time of the adenyl derivatives reveal that the adenylyl site is located on the surface of the enzyme. 

The main source of conformational information for biopolymers are the easy-to-obtain chemical shifts that can be translated into dihedral restraints. In addition, for fully 13C labeled compounds, proton-driven spin diffusion between carbons [72] can be used to measure quantitatively distances between carbons. The CHHC experiment is the equivalent of the NOESY in solution that measures distances between protons by detecting the resonances of the attached carbons. While both techniques, proton-driven spin diffusion and CHHC experiment [73], allow for some variation in the distance as determined from cross-peak integrals, REDOR [74] experiments in selective labeled compounds measure very accurate distances by direct observation of the oscillation of a signal by the dipolar coupling. While the latter technique provides very accurate distances, it provides only one piece of information per sample. Therefore, the more powerful techniques proton-driven spin diffusion and CHHC have taken over when it comes to structure determination by ss-NMR of fully labeled ligands. 

Simultaneously with the refinement of the EC structure, additional data on the conformation of taxol bound to MT came from ss-NMR REDOR measurements [84]. As introduced above, the REDOR experiment exploits the dipolar interaction between nuclear spins to determine distances between atoms closer than 12 A. A PTX analogue with suitable spin labeling for REDOR was prepared and complexed to MT. Labeling of the amide carbonyl and C3 methine with 13C, and position p- of the 2-O-benzoyl with 19F (Fig. 9, compound 3), allowed the measurement of the two 19F-13C distances I and II (Fig. 9), that could serve, in principle, to discriminate among candidates for PTX conformation. The respective values of 9.8 A 0.5 A and 10.3 A 0.5 A were interpreted by the authors as supportive of the hydrophobi-cally collapsed polar conformation for PTX [84]. 

Kulikov, A.V. (1976) Determination of distance between the nitroxide label and paramagnetic center in spin-labeled proteins from the parameters of the saturation curve of the ESR spectrum of the label at 77K. Molecul. Biol. (Moscow) 10, 109-116. 

Rabenstein, M. D., and Shin, Y K. (1995). Determination of the distance between two spin labels attached to a macromolecule. Proc. Natl. Acad. Sci. USA 92, 8239-8243. 

Further determinations of intramolecular distances have been made by Musci et al. (1987). Met-90 in a-lactalbumin was spin-labeled. Paramagnetic line broadening of the spin-laheled ESR lines by Gd(III), substituted at the high-affinity site, yielded a distance of 8 1 A between the spin label and the metal binding site. Distances between the Met and several resolvable protons were also determined from paramagnetic line broadening, with the use of NMR. 

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