The Characterization of Weak Protein–Protein Interactions: Evidence from DEER for the Trimerization of a von Willebrand Factor A Domain in Solution

Banham, Janet E.; Timmel, Christiane R.; Abbott, Rachel J. M.; Lea, Susan M.; Jeschke, Gunnar

doi:10.1002/anie.200503720

Cited by 64 publications

(51 citation statements)

References 16 publications

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“…Pulsed electron-electron double resonance (PELDOR) experiments for the determination of distances between paramagnetic centers [l, 2] have recently become a very active field of research with numerous applications on nanostructured materials [3][4][5][6][7][8], soluble proteins [9][10][11][12][13][14][15][16][17], membrane proteins [18][19][20][21], nucleic acids [22], and peptides [23]. In many of these studies, careful analysis of the dipolar evolution data was recognized asa crucial precondition for their reliable interpretation.…”

Section: Introductionmentioning

confidence: 99%

DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

et al. 2006

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Pulsed electron-electron double resonance techniques such as the four-pulse double electron-electron resonance experiment measure a dipolar evolution function of the sample. For a sample consisting of spin-carrying nanoobjects, this function is the product of a form factor, corresponding to the internal structure of the nanoobject, and a background factor, corresponding to the distribution of nanoobjects in space. The form factor contains information on the spin-to-spin distance distribution within the nanoobject and on the average number of spins per nanoobject, while the background factor depends on constraints, such as a confinement of the nanoobjects to a two-dimensional layer. Separation of the dipolar evolution function into these two contributions and extraction of the spinto-spin distance distribution require numerically stable mathematical algorithms that can handle data for different classes of samples, e.g., spin-labelled biomacromolecules and synthetic materials. Furthermore, experimental imperfections such as the limited excitation bandwidth of microwave pulses need to be considered. The software package DeerAnalysis2006 provides access to a comprehensive set of tools for such data analysis within a common user interface. This interface allows for several tests of the reliability and precision of the extracted information. User-supplied models for the spinto-spin distance distribution within a certain class of nanoobjects can be added to an existing library and be fitted with a universal algorithm.

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Section: Introductionmentioning

confidence: 99%

DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

et al. 2006

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“…The perturbation causes a change in the dipolar field of the detected spin and results in a modulation of the time-domain spin echo signal as a function of the dipolar frequency. At low EPR frequencies (X band), this method has been successfully applied in structural biology to measure distances between native cofactors in proteins, such as those of photosystem II (13), hydrogenase (14), RNR (10,15,16), sulfhydryl oxidases (17), and pyruvate ferredoxin oxidoreductase (18) or, by using nitroxide spin labels, to determine distances in RNA (19), DNA (20), and membrane proteins (21,22). A very early study by Larsen and Singel (23) investigated the effect of orientation dependence in PELDOR at low fields using nitroxide spin labels.…”

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confidence: 99%

High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

Denysenkov

Prisner

Stubbe

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

142

117

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Class I ribonucleotide reductases (RNRs) are composed of two subunits, R1 and R2. The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2-deoxynucleoside diphosphates. It has been proposed that the function of the tyrosyl radical in R2 is to generate a transient thiyl radical (C439⅐) in R1 over a distance of 35 Å, which in turn initiates the reduction process. EPR distance measurements provide a tool with which to study the mechanism of radical initiation in class I RNRs. These types of experiments at low magnetic fields and frequencies (0.3 T, 9 GHz) give insight into interradical distances and populations. We present a pulsed electron-electron double resonance (PELDOR) experiment at high EPR frequency (180-GHz electron Larmor frequency) that detects the dipolar interaction between the Y⅐s in each protomer of RNR R2 from Escherichia coli. We observe a correlation between the orientation-dependent dipolar interaction and their resolved g-tensors. This information has allowed us to define the relative orientation of two radicals embedded in the active homodimeric protein in solution. This experiment demonstrates that high-field PELDOR spectroscopy is a powerful tool with which to study the assembly of proteins that contain multiple paramagnetic centers.double electron-electron resonance ͉ distance measurements R ibonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. Class I RNR from Escherichia coli is composed of two homodimeric subunits (R1 and R2) that are thought to form a 1:1 complex (1). The R2 subunit contains the essential diferric cluster-tyrosyl radical (Y⅐) cofactor, and R1 is the site of the conversion of nucleoside diphosphates to 2Ј-deoxynucleoside diphosphates. The chemistry of nucleotide reduction is moderately well understood (2), and structures of R1 (3) and R2 (4, 5), as well as a recent structure of the R1:R2 holocomplex (6), are available. A major unresolved issue in this class of enzymes is the mechanism of radical initiation (7): How does the tyrosyl radical in R2 generate a transient thiyl radical in R1 over a distance of 35 Å? The current proposal for the radical propagation pathway is based on a docking model of R1 and R2 and involves aromatic amino acid residues (3,8,9). Evidence in support of the long distance and the docking model has been recently provided by pulsed EPR distance measurements (10). These experiments have detected the distance between the Y⅐ in R2 and a nitrogencentered radical in the active site of R1, providing structural information on the R1:R2 active complex in the presence of the substrate and the allosteric effector. The results provided the impetus to further explore the capability of the method at high magnetic fields.The pulsed electron-electron double resonance (PELDOR) experiment detects weak dipolar interactions between radicals and is based on a two-frequency pulse sequence (11, 12). One frequency is requ...

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“…In Monte Carlo simulations of homogeneous distributions either only the distance (test of Eqs. (13) and (14)) or the distance and angles h g between the magnetic field and the unique axis of the Dy 3+ g tensor and h between the magnetic field and the spin-spin vector were varied.…”

Section: Monte Carlo Simulationsmentioning

confidence: 99%

“…With commercial hardware and without isotope labeling such methods can routinely access the distance range between 1.8 and 5 nm, which fits well to the size of the target structures. Double-quantum techniques combined with dedicated hardware [4] may allow for an extension to somewhat shorter distances while deuterium exchange of the matrix allows for an extension to longer distances [11][12][13], in favorable cases up to 8 nm [14]. Good-quality measurements can be performed on samples with spin label concentrations down to about 50 lM.…”

Section: Introductionmentioning

confidence: 99%

Relaxation-based distance measurements between a nitroxide and a lanthanide spin label

Jäger

Koch

Maus

et al. 2008

Journal of Magnetic Resonance

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The Characterization of Weak Protein–Protein Interactions: Evidence from DEER for the Trimerization of a von Willebrand Factor A Domain in Solution

Cited by 64 publications

References 16 publications

DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

DeerAnalysis2006—a comprehensive software package for analyzing pulsed ELDOR data

High-field pulsed electron–electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase

Relaxation-based distance measurements between a nitroxide and a lanthanide spin label

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