Measuring Cu2+-Nitroxide Distances Using Double Electron–Electron Resonance and Saturation Recovery

Sarver, Jessica; Silva, K. Ishara; Saxena, Sunil

doi:10.1007/s00723-012-0422-x

Cited by 17 publications

(26 citation statements)

References 46 publications

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“…52,64 The stepsize was 16 ns in all DEER measurements with a total of 110 to 150 points. The pump and the observe pulses were applied to the maximum absorbance of the nitroxide and the Cu 2+ absorbance spectrum, respectively.…”

Section: Methodsmentioning

confidence: 99%

Long-Range Distance Measurements in Proteins at Physiological Temperatures Using Saturation Recovery EPR Spectroscopy

et al. 2014

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Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron–electron resonance (DEER), is carried out at cryogenic temperatures (50–80 K) to increase the short spin–spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20–60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin–lattice relaxation rate (T1–1) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu2+, it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu2+ binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu2+ electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu2+ loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu2+/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.

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Section: Methodsmentioning

confidence: 99%

Long-Range Distance Measurements in Proteins at Physiological Temperatures Using Saturation Recovery EPR Spectroscopy

et al. 2014

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“…Cu 2þ ions offer an alternative spin probe (9) that for Cu-metalloproteins requires no perturbation of the sample (10). Moreover, when Cu 2þ and nitroxide labels are combined in a single sample, triangulation of distance restraints (11,12) can provide a more global view of structure and dynamics (13). Here, we apply Cu-PDS to the enzyme Cu-Zn superoxide dismutase (SOD1, Fig.…”

Section: Introductionmentioning

confidence: 99%

Copper-Based Pulsed Dipolar ESR Spectroscopy as a Probe of Protein Conformation Linked to Disease States

Merz

Borbat

Pratt

et al. 2014

Biophysical Journal

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We demonstrate the ability of pulsed dipolar electron spin resonance (ESR) spectroscopy (PDS) to report on the conformation of Cu-Zn superoxide dismutase (SOD1) through the sensitive measurement of dipolar interactions between inherent Cu(2+) ions. Although the extent and the anisotropy of the Cu ESR spectrum provides challenges for PDS, Ku-band (17.3 GHz) double electron-electron resonance and double-quantum coherence variants of PDS coupled with distance reconstruction methods recover Cu-Cu distances in good agreement with crystal structures. Moreover, Cu-PDS measurements expose distinct differences between the conformational properties of wild-type SOD1 and a single-residue variant (I149T) that leads to the disease amyotrophic lateral sclerosis (ALS). The I149T protein displays a broader Cu-Cu distance distribution within the SOD1 dimer compared to wild-type. In a nitroxide (NO)-labeled sample, distance distributions obtained from Cu-Cu, Cu-NO, and NO-NO separations reveal increased structural heterogeneity within the protein and a tendency for mutant dimers to associate. In contrast, perturbations caused by the ALS mutation are completely masked in the crystal structure of I149T. Thus, PDS readily detects alterations in metalloenzyme solution properties not easily deciphered by other methods and in doing so supports the notion that increased range of motion and associations of SOD1 ALS variants contribute to disease progression.

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“…Although this approach is quick and straightforward, it has several disadvantages: 1) it is difficult to read of the frequencies of the singularities in the case of broad distance distributions, 2) only the mean inter‐spin distance but not its distribution can be determined, and 3) orientation selectivity may lead to a distribution P ( θ ), in which the most probable value does not correspond to θ =90°. These issues were circumvented by taking orientation selectivity explicitly into account in the PELDOR data analysis . This was done in the following way: In order to gather sufficient information about the orientation selectivity, several PELDOR time traces were recorded with different pump/detection positions across the Cu 2+ spectrum.…”

Section: Methodsmentioning

confidence: 99%

“…These issues were circumvented by taking orientation selectivity explicitly into account in the PELDOR data analysis. [44,56,[60][61][62][63][64][65][66] This was done in the following way: In order to gather sufficient information about the orientation selectivity, several PELDOR time traces were recorded with different pump/detection positions across the Cu 2 + spectrum. Next, these PELDOR time traces were simulated all together using a geometric model, which includes the inter-spin distance and orientation of the spin centers.…”

Section: Methodology Copper(ii)mentioning

confidence: 99%

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

Abdullin

Schiemann

2020

ChemPlusChem

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Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) is a valuable method for determining biomolecular structures and their conformational changes during function. Often, this method is applied to paramagnetic metal ions that are either intrinsic co‐factors of biomolecules or introduced into biomolecules as spin labels. Here, the key achievements and the remaining challenges of PDS on metal ions are summarized and critically discussed. The first part of the Review describes the methodology of PDS experiments and the related data analysis for each of the biologically relevant paramagnetic metal centers. The second part highlights applications with the aim to give an idea about what kind of questions from structural biology can be answered by PDS‐based distance measurements involving metal centers.

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Measuring Cu2+-Nitroxide Distances Using Double Electron–Electron Resonance and Saturation Recovery

Cited by 17 publications

References 46 publications

Long-Range Distance Measurements in Proteins at Physiological Temperatures Using Saturation Recovery EPR Spectroscopy

Long-Range Distance Measurements in Proteins at Physiological Temperatures Using Saturation Recovery EPR Spectroscopy

Copper-Based Pulsed Dipolar ESR Spectroscopy as a Probe of Protein Conformation Linked to Disease States

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

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