Analyzing CW EPR Spectra of Nitroxide Labeled Macromolecules

Altenbach, Christian; Budil, David

doi:10.1007/s00723-023-01610-2

Cited by 6 publications

(2 citation statements)

References 73 publications

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“…2E) were fit to a 3-component system (Fig. S4) using the MultiComponent software (39), (see Methods). The spectra were assumed to be composed of 3 major sub-populations identified in earlier studies (40): (i) a mobile component describing soluble monomers, (ii) an immobile component describing labeled peptides embedded in a fibril that is tumbling slowly, and (iii) a dipolar and/or spin-exchange broadened component describing spin-labels in a fibril within close proximity (< 15 Å) of another label (Fig.…”

Section: P301l Mutation Of Jr2r3 Results In a Greater Population Of S...mentioning

confidence: 99%

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

Vigers,

Lobo,

Najafi

et al. 2023

Preprint

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Tau forms toxic fibrillar aggregates in a family of neurodegenerative diseases known as tauopathies. The faithful replication of tauopathy-specific fibril structures is a critical gap for developing diagnostic and therapeutic tools. This study debuts a strategy of identifying a critical segment of tau that forms a folding motif that is characteristic of a family of tauopathies and isolating it as a standalone peptide that form seeding-competent fibrils. The 19-residue jR2R3 peptide (295-313) spanning the R2/R3 splice junction of tau, in the presence of P301L, forms seeding-competent amyloid fibrils. This tau fragment contains the hydrophobic VQIVYK hexapeptide that is part of the core of every pathological tau fibril structure solved to-date and an intramolecular counter-strand that stabilizes the strand-loop-strand (SLS) motif observed in 4R tauopathy fibrils. This study shows that P301L exhibits a duality of effects: it lowers the barrier for the peptide to adopt aggregation-prone conformations and enhances the local structuring of water around the mutation site that facilitates site-specific dewetting and in-register stacking of tau to form cross β-sheets. We solve a 3 Å cryo-EM structure of jR2R3-P301L fibrils with a pseudo 21screw symmetry in which each half of the fibril’s cross-section contains two jR2R3-P301L peptides. One chain adopts a SLS fold found in 4R tauopathies that is stabilized by a second chain wrapping around the SLS fold, reminiscent of the 3-fold and 4-fold structures observed in 4R tauopathies. These jR2R3-P301L fibrils are able to template full length tau in a prion-like fashion.Significance StatementThis study presents a first step towards designing a tauopathy specific aggregation pathway by engineering a minimal tau prion building block, jR2R3, that can template and propagate distinct disease folds. We present the discovery that P301L—among the widest used mutations in cell and animal models of Alzheimer’s Disease—destabilizes an aggregation-prohibiting internal hairpin and enhances the local surface water structure that serves as an entropic hotspot to exert a hyper-localized effect in jR2R3. Our study suggests that P301L may be a more suitable mutation to include in modeling 4R tauopathies than for modelling Alzheimer’s Disease, and that mutations are powerful tools for the purpose of designing of tau prion models as therapeutic tools.

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Section: P301l Mutation Of Jr2r3 Results In a Greater Population Of S...mentioning

confidence: 99%

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

Vigers,

Lobo,

Najafi

et al. 2023

Preprint

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show abstract

“…Variable-pressure CW EPR data were amplitude normalized and background corrected using custom software Convert&Align and Baseline, respectively (written in LabVIEW and available upon request). The pressure dependent CW EPR spectra were simulated with MultiComponent (Altenbach & Budil, 2023), which utilizes theory developed by Freed and coworkers (Budil et al, 1996) to extract dynamic parameters as well as populations to determine the apparent equilibrium constants (K) (Table S2).…”

Section: Cw Spectral Simulation and Calculation Of Thermodynamic Para...mentioning

confidence: 99%

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

Grosskopf,

Sidabras,

Altenbach

et al. 2024

Preprint

Self Cite

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Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-us using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of us-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

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A pressure‐jump EPR system to monitor millisecond conformational exchange rates of spin‐labeled proteins

Grosskopf,

Sidabras,

Altenbach

et al. 2024

Protein Science

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Site‐directed spin labeling electron paramagnetic resonance (SDSL‐EPR) using nitroxide spin labels is a well‐established technology for mapping site‐specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL‐EPR also provides information on protein dynamics in the timescale of ps–μs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of μs–ms, corresponding to large‐scale protein motions, is inaccessible to those methods. To extend SDSL‐EPR to the longer time domain, the perturbation method of pressure‐jump relaxation is implemented. Here, we describe a complete high‐pressure EPR system at Q‐band for both static pressure and ms‐timescale pressure‐jump measurements on spin‐labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding–unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the ms timescale.

show abstract

Analyzing CW EPR Spectra of Nitroxide Labeled Macromolecules

Cited by 6 publications

References 73 publications

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

Tau P301L mutation promotes core 4R tauopathy fibril fold through near-surface water structuring and conformational rearrangement

A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

A pressure‐jump EPR system to monitor millisecond conformational exchange rates of spin‐labeled proteins

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