Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Banham, Janet E.; Baker, Christopher M.; Ceola, Stefano; Day, Iain J.; Grant, Guy H.; Groenen, E. J. J.; Rodgers, Christopher T.; Jeschke, Gunnar; Timmel, Christiane R.

doi:10.1016/j.jmr.2007.11.023

Cited by 144 publications

(123 citation statements)

References 35 publications

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“…The envelope of the best two Gaussian fit are plotted in each case using dashed lines. The average distances and standard deviations extracted from the fits are compiled in Tables 2 and 3. of relatively low signal-to-noise and the difficulty of obtaining accurate distances below 2 nm using DEER measurements (29,35,45). The fact that the presence of these shorter distance components in the data appears to be random with respect to protein variant or sample conditions supports our conclusion that they are artifacts.…”

Section: Inter-spin Distances In Membrane-bound ␣Ssupporting

confidence: 68%

“…Although the flexibility of the nitroxide spin labels is expected to introduce a level of uncertainty into the measurement, this effect is not expected to be larger than ϳ1 nm (34,(45)(46)(47). For measurements of the very long distances associated with the extended helix conformation, long signal acquisition times are required, leading to a decreased signal-to-noise ratio and potentially contributing to a larger uncertainty in the distance reconstruction algorithm (34).…”

Section: Figure 4 -Continuedmentioning

confidence: 99%

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The Lipid-binding Domain of Wild Type and Mutant α-Synuclein

Georgieva

Ramlall

Borbat

et al. 2010

Journal of Biological Chemistry

134

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␣-Synuclein (␣S) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three ␣S point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of ␣S is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, ␣S undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of ␣S likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent-and membrane-bound conformations of ␣S, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked ␣S mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.Since its initial discovery as a protein enriched in the electric organ of the Pacific electric ray, Torpedo californica (1), the protein ␣-synuclein has been progressively associated with a role in neurodegeneration in humans, starting with its identification as the precursor protein of a non-amyloid-␤ peptide component of amyloid plaques (the so-called NAC peptide) (2) and culminating in its discovery as the first gene product to be linked to familial Parkinson disease (3) and its contemporaneous identification as the primary component of the fibrillary aggregates found in the hallmark Lewy body deposits associated with Parkinson (4).Subsequently, a great deal of effort has been made to clarify and understand the mechanisms by which ␣S causes neuronal degeneration, as well as to understand the normal functional roles of ␣S and how these are related to its role in disease (5, 6). Although aggregation of ␣S remains a key focus in these efforts, it remains possible that other, or additional, effects beyond the presence of various aggregated species of ␣S may be involved in the etiology of synuclein-associated PD. A key piece of the puzzle that remains unanswered is which of the various forms of ␣S that have been observed in vitro are involved in mediating the toxicity of the protein in vivo. In particular, although ␣S is highly disordered when isolated in solution in vitro (7-9) it is not clear to what extent this intrinsically disordered state is populated in vivo. Be...

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Section: Inter-spin Distances In Membrane-bound ␣Ssupporting

confidence: 68%

Section: Figure 4 -Continuedmentioning

confidence: 99%

The Lipid-binding Domain of Wild Type and Mutant α-Synuclein

Georgieva

Ramlall

Borbat

et al. 2010

Journal of Biological Chemistry

134

View full text Add to dashboard Cite

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“…Figs. 5 and 6 confirm that CW EPR and DEER are quite complementary techniques for determining distance distributions (28). For all cases, CW EPR and DEER data were consistent, but CW EPR was superior in sensitivity and resolution for distances of Ͻ2 nm (Fig.…”

Section: Sdsl-epr Methodology and Computationalsupporting

confidence: 58%

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

Klein

Burr

Svensson

et al. 2008

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

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We present a structurally dynamic model for nucleotide-and actin-induced closure of the actin-binding cleft of myosin, based on site-directed spin labeling and electron paramagnetic resonance (EPR) in Dictyostelium myosin II. The actin-binding cleft is a solventfilled cavity that extends to the nucleotide-binding pocket and has been predicted to close upon strong actin binding. Single-cysteine labeling sites were engineered to probe mobility and accessibility within the cleft. Addition of ADP and vanadate, which traps the posthydrolysis biochemical state, influenced probe mobility and accessibility slightly, whereas actin binding caused more dramatic changes in accessibility, consistent with cleft closure. We engineered five pairs of cysteine labeling sites to straddle the cleft, each pair having one label on the upper 50-kDa domain and one on the lower 50-kDa domain. Distances between spin-labeled sites were determined from the resulting spin-spin interactions, as measured by continuous wave EPR for distances of 0.7-2 nm or pulsed EPR (double electron-electron resonance) for distances of 1.7-6 nm. Because of the high distance resolution of EPR, at least two distinct structural states of the cleft were resolved. Each of the biochemical states tested (prehydrolysis, posthydrolysis, and rigor), reflects a mixture of these structural states, indicating that the coupling between biochemical and structural states is not rigid. The resulting model is much more dynamic than previously envisioned, with both open and closed conformations of the cleft interconverting, even in the rigor actomyosin complex.double electron-electron resonance ͉ dipolar interaction ͉ actomyosin M yosin motors use the energy derived from ATP hydrolysis to generate force for a diverse repertoire of biological tasks (1). Class II myosins produce muscle contraction and are involved in cytokinesis and cell motility. The present study focuses on Dictyostelium discoideum (Dicty) myosin II, which offers optimal facility for expression and purification of mutants, and is functionally similar to muscle myosin II (2, 3).

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“…To test for the possibility that Syt1 is oligomerized in the bilayer, we examined both single and double spin-labeled linker mutants using double electron-electron resonance (DEER). If pairs of nitroxides are in proximity at defined distances, this pulse EPR measure- ment produces a signal that is modulated at the frequency of the dipolar interaction (45). Shown in Fig.…”

Section: Reconstitution and Vesicle Capture By Synaptotagmin 1-mentioning

confidence: 99%

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

Lü

Kiessling

Tamm

et al. 2014

Journal of Biological Chemistry

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Background: Synaptotagmin 1 is the Ca 2ϩ sensor for neuronal exocytosis. Results: The juxtamembrane linker of synaptotagmin 1 is oligomerized through a glycine zipper motif, and this interaction controls the activity of synaptotagmin 1. Conclusion: Structural modifications in the linker alter the activity of synaptotagmin 1. Significance: The oligomerization of synaptotagmin 1 may control the organization at the focal site of fusion.

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Distance measurements in the borderline region of applicability of CW EPR and DEER: A model study on a homologous series of spin-labelled peptides

Cited by 144 publications

References 35 publications

The Lipid-binding Domain of Wild Type and Mutant α-Synuclein

The Lipid-binding Domain of Wild Type and Mutant α-Synuclein

Actin-binding cleft closure in myosin II probed by site-directed spin labeling and pulsed EPR

The Juxtamembrane Linker of Full-length Synaptotagmin 1 Controls Oligomerization and Calcium-dependent Membrane Binding

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