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

Merz, Gregory E.; Borbat, Peter P.; Pratt, Ashley J.; Getzoff, Elizabeth D.; Freed, Jack H.; Crane, Brian R.

doi:10.1016/j.bpj.2014.07.068

Cited by 35 publications

(32 citation statements)

References 56 publications

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“…We noted differences in the overall intersubunit peak breadth for several G93 mutants (Fig. 6 C and D), and related work has suggested that this feature is common to additional ALS mutants (98). Conceivably, this flexibility could enable increased conformational sampling, rendering mutant SOD proteins more susceptible to metal loss and aggregation.…”

Section: +supporting

confidence: 51%

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Pratt

Shin

Merz³

et al. 2014

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

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Protein framework alterations in heritable Cu, Zn superoxide dismutase (SOD) mutants cause misassembly and aggregation in cells affected by the motor neuron disease ALS. However, the mechanistic relationship between superoxide dismutase 1 (SOD1) mutations and human disease is controversial, with many hypotheses postulated for the propensity of specific SOD mutants to cause ALS. Here, we experimentally identify distinguishing attributes of ALS mutant SOD proteins that correlate with clinical severity by applying solution biophysical techniques to six ALS mutants at human SOD hotspot glycine 93. A small-angle X-ray scattering (SAXS) assay and other structural methods assessed aggregation propensity by defining the size and shape of fibrillar SOD aggregates after mild biochemical perturbations. Inductively coupled plasma MS quantified metal ion binding stoichiometry, and pulsed dipolar ESR spectroscopy evaluated the Cu 2+ binding site and defined cross-dimer copper-copper distance distributions. Importantly, we find that copper deficiency in these mutants promotes aggregation in a manner strikingly consistent with their clinical severities. G93 mutants seem to properly incorporate metal ions under physiological conditions when assisted by the copper chaperone but release copper under destabilizing conditions more readily than the WT enzyme. Altered intradimer flexibility in ALS mutants may cause differential metal retention and promote distinct aggregation trends observed for mutant proteins in vitro and in ALS patients. Combined biophysical and structural results test and link copper retention to the framework destabilization hypothesis as a unifying general mechanism for both SOD aggregation and ALS disease progression, with implications for disease severity and therapeutic intervention strategies. Lou Gehrig's disease | small-angle X-ray scattering | protein aggregation | protein conformation | ESR spectroscopy A LS is a lethal degenerative disease of the human motor system (1). Opportunities for improved understanding and clinical intervention arose from the discovery that up to 23.5% of familial ALS cases and 7% of spontaneous cases are caused by mutations in the superoxide dismutase 1 (SOD1) gene encoding human Cu, Zn SOD (2-4). SOD is a highly conserved (5), dimeric, antioxidant metalloenzyme that detoxifies superoxide radicals (6, 7), but overexpression of SOD1 ALS mutants is sufficient to cause disease in mice (8). Misfolded and/or aggregated SOD species are deposited within mouse neuronal and glial inclusions (9, 10), even before symptoms appear (11,12). Although human familial ALS has a symptomatic phenotype indistinguishable from sporadic cases (13), individual SOD1 mutations can result in highly variable disease progression and penetrance (14,15).Many nongeneral mechanisms, including loss of activity or gain of function, were postulated to explain the roles of SOD mutants in ALS (3,(16)(17)(18)(19). Recently, however, an initial hypothesis proposing that SOD manifests disease symptoms by framework dest...

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Section: +supporting

confidence: 51%

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Pratt

Shin

Merz³

et al. 2014

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

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“…However, DEER and DQC experiments on metal ions are challenging given the lower sensitivity. Nevertheless, our group [26,27,29,30] and others [17,23,[31][32][33][34][35][36][37] have made tangible progress. DEER has significant advantages for Cu 2+ based experiments given that nuclear interactions are easier to reduce and due to the ease of implementation on commercial instruments.…”

Section: Introductionmentioning

confidence: 95%

“…Two techniques have been developed for the 12-100 Å distance range: double electron-electron resonance (DEER) [10,11] and double quantum coherence (DQC) [12,13]. In the DEER experiments, the local dipolar field due to the coupled spin-partner is inverted using a second mw channel, and the duration of the pulses is usually longer than 8 ns [14][15][16][17]. The echo is then modulated by the dipolar frequency providing sensitivity to distances.…”

Section: Introductionmentioning

confidence: 99%

A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements

Ruthstein

Shin

et al. 2015

Journal of Magnetic Resonance

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Double quantum coherence (DQC) ESR is a sensitive method to measure magnetic dipolar interactions between spin labels. However, the DQC experiment on Cu(2+) centers presents a challenge at X-band. The Cu(2+) centers are usually coordinated to histidine residues in proteins. The electron-nuclear interaction between the Cu(2+) ion and the remote nitrogen in the imidazole ring can interfere with the electron-electron dipolar interaction. Herein, we report on a modified DQC experiment that has the advantage of reduced contributions from electron-nuclear interactions, which enhances the resolution of the DQC signal to the electron-electron dipolar modulations. The modified pulse-sequence is verified on Cu(2+)-NO system in a polyalanine-based peptide and on a coupled Cu(2+) system in a polyproline-based peptide. The modified DQC data were compared with the DEER data and good agreement was found.

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“…However, DQC pulse sequences have been developed which appear to reduce the electron-nuclear interaction, and may allow the sensitivity of DQC methods to be exploited for distance measurements [53]. Recently, both DEER and DQC methods have been used to show that mutations in the superoxide dismutase 1 (CuZnSOD) resulted in structural heterogeneity, which may be a contributing factor in the development of amyotrophic lateral sclerosis [54].…”

Section: Using Epr To Determine a Copper(ii)-nitroxide Distancementioning

confidence: 99%

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function

Jones

Berliner

2016

Cell Biochem Biophys

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Copper is one of the most abundant biological metals, and its chemical properties mean that organisms need sophisticated and multilayer mechanisms in place to maintain homoeostasis and avoid deleterious effects. Studying copper proteins requires multiple techniques, but electron paramagnetic resonance (EPR) plays a key role in understanding Cu(II) sites in proteins. When spin-labels such as aminoxyl radicals (commonly referred to as nitroxides) are introduced, then EPR becomes a powerful technique to monitor not only the coordination environment, but also to obtain structural information that is often not readily available from other techniques. This information can contribute to explaining how cuproproteins fold and misfold. The theory and practice of EPR can be daunting to the non-expert; therefore, in this mini review, we explore how nitroxide spin-labelling can be used to help the inorganic biochemist gain greater understanding of cuproprotein structure and function in vitro and how EPR imaging may help improve understanding of copper homoeostasis in vivo.

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

Cited by 35 publications

References 56 publications

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

Aggregation propensities of superoxide dismutase G93 hotspot mutants mirror ALS clinical phenotypes

A simple double quantum coherence ESR sequence that minimizes nuclear modulations in Cu2+-ion based distance measurements

Nitroxide Spin-Labelling and Its Role in Elucidating Cuproprotein Structure and Function

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