Interspin distances

The relaxation enhancement displayed for canthaxanthin, 7 -apo-7 -(4-carboxyphenyl)-P-carotene and BI was analyzed to provide interspin distances. The dipolar interactions and the distances can be determined according to procedures described elsewhere (Budker et al. 1995, Rakowsky et al. 1995, Eaton and Eaton 2000, Rao et al. 2000) and based on simulations of the paramagnetic metal ion contribution, WAA. 

D is the dipole-dipole interaction between the slow relaxing carotenoid radical and the fast relaxing Ti3+ ion r is the interspin distance... 

The cross-relaxation rates between two spins can be experimentally measured in the laboratory (rotating frame They depend on interspin distance r and correlation time that modulates the dipole-dipole interaction [4] ... 

Analytical expressions were derived for the CW EPR line-shape for spin-polarized radical pairs in the limit where the combined dipolar and exchange interaction is weak relative to the energy differences between the resonances of the two spins.19 The equations were applied to the case of charge-separated sites in Ti02 nanoparticles. This approach simplifies the analysis of the distributions of interspin distances. 

Pulse Measurements. Comparison of 3-pulse PELDOR data for nitroxide biradicals with interspin distances of 15.4 to 24.0 A at X-band and S-band demonstrated the separation of nuclear hyperfine and dipolar interactions and the separation of contributions from dipolar and exchange interactions.20 However, the deeper nuclear modulation at S-band limits the options for interpulse spacings. The inability to observe dipolar modulation in a PELDOR experiment... 

The four-pulse DEER experiment permits dead-time free measurements.21 For a nitroxide biradical with an interspin distance of 19.4 A there was good agreement between results obtained by 3-pulse and 4-pulse DEER. For a second biradical the interspin distance of 28.3 A obtained by 4-pulse DEER was in good agreement with the X-ray crystal structure. The accessible distance range was estimated to be 15 to 80 A. 

Double-quantum coherence (DQC) was used to measure interspin distances in eight doubly labelled derivatives of T4 lysozyme.25 Distances between 20 and 47 A with distance distributions of 1.0 to 2.8 A were measured. For some variants two conformations were detected with distances that differed by 5 to 5.4 A. It was shown that a few measurements of large distances provided significant constraints on protein structure. 

Rapid stepping of the magnetic field, instead of using a second microwave frequency, has been used to measure interspin distances of the order of 20 A at X-band.28 A microwave pulse, called the pump pulse, is synchronized with the field step and occurs between the second and third pulses of a stimulated echo sequence. The effects of nuclear ESEEM were removed by dividing data obtained with a pump pulse (at the stepped magnetic field) by data obtained without a pump pulse. 

Pairs of W5+ ions were observed in lithium-tungsten phosphate glasses.35 The effectiveness of pair formation increased as the Li+ concentration increased. Interspin distances were calculated from the dipolar splitting, D. The mean distance between W5+ ions decreased from 4.53 A for the glass without Li+ ions to 3.70 A for glasses containing 50 mol% Li+. 

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. 

Three doubly spin-labelled [2]catenanes with different sizes were studied by 4-pulse DEER.52 The experimental distribution of interspin distances was compared with a theoretical pair-correlation function computed based on geometrical constraints. In chloroform solution the medium and large catenanes were close to fully expanded, but in glassy o-terphenyl they were partially collapsed. For the smaller catenane there was a higher population of shorter interspin distances, which was attributed to interactions between unsaturated sections of the molecule. 

Applications to Biological Samples. - Methods of distance measurements were compared for four doubly spin-labelled derivatives of human carbonic anhydrase.53 The distances between the spin labels were obtained from continuous wave spectra by analysis of the relative intensity of the half-field transition, Fourier deconvolution of the line-shape broadening, and computer simulation of line-shape changes. For variants with interspin distances greater than 18 A, the DEER method also was used. For each variant, at least two methods were applicable and reasonable agreement between distances obtained by different methods was obtained. The useful distance ranges for the techniques employed at X-band with natural isotope abundance spin labels were estimated to be half-field transition (5-10 A), line-shape simulation (up to 15 A), Fourier deconvolution (8 - 20 A), and four-pulse DEER (> 18 A).53... 

CW line-shape simulation. Simulation of the EPR spectrum of di-copper-substituted aminopeptidase from Aeromonas proteolytica found a zero field splitting that corresponded to an interspin distance of about 5 A, which is in good agreement with the X-ray crystallographic value of 4.93 A.55... 

A spin label was attached by photoaffinity to the nucleotide binding site of sarcoplasmic reticulum Ca2+-ATPase.56 Dipolar interaction corresponding to an interspin distance of about 15 A was observed, which demonstrated that the minimal functional unit is a dimer with the nucleotide-binding sites of the two monomers in close proximity. 

Archaebacterial rhodopsins may provide models for signal transduction and ion transport.62 Spin labels were attached to sensory rhodopsin (pSRII) and its transducer (pHTrll) from Natronobacterium pharaonis. Interspin distance determined by line-shape analysis including a Gaussian distribution of distances revealed a 2 2 complex with 2-fold symmetry. Distances for 26 pairs of spin labels defined the orientation of the TM helices of pHtrll relative to the F and G helices of pSRII.63 Light excitation causes a flap-like movement of helix F of NpSRII that induces a rotary motion of a helix in the transmembrane domain of the transducer.62... 

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. 

Pairs of spin labels were introduced in human prostaglandin endoperoxide H2 synthase-2 (PGHS-2) at locations where significant structural changes had been observed in X-ray crystal structures of holoenzyme and protein bound to NSAID s.75 Interspin distances were obtained by Fourier deconvolution of spectra recorded at 183 K for 10 doubly spin-labelled mutants. Interspin distances for complexes with arachidonic acid, flurbiprofen, and SC-58125 agree well with values predicted from X-ray crystal structures, but do not agree well for the holoenzyme. It is proposed that the predominant conformation of the holoenzyme in solution is different from the structure in the crystal. 

Pulse measurements. A self-complementary RNA with 12 base pairs was spin labelled at uridine near the middle of the strand.83 Three-pulse PELDOR gave an interspin distance of 35 2 A, which is in good agreement with molecular modelling. For a shorter self-complementary RNA, dipolar modulation was not observed, which was attributed to aggregation or weak duplex formation. 

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. 

Two R1 side chains in a protein, or an R1 side chain and a paramagnetic metal ion in an engineered site, interact by a distance-dependent magnetic dipolar interaction. For the usual case where there is a distribution of interspin distances, the dipolar interaction leads to an EPR... 

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