Structural context modulates the conformational ensemble of the intrinsically disordered amino terminus of α-synuclein.

Regions of intrinsic disorder play crucial roles in biological systems, yet they often elude characterization by conventional biophysical techniques. To capture conformational distributions across different timescales, we employed a freezing approach coupled with solid-state NMR analysis. Using segmentally isotopically labeled α-synuclein (α-syn), we investigated the conformational preferences of the six alanines, three glycines, and a single site (L8) in the disordered amino terminus under three distinct conditions: in 8 M urea, as a frozen monomer in buffer, and within the disordered regions flanking the amyloid core.

In cell NMR reveals cells selectively amplify and structurally remodel amyloid fibrils.

Amyloid forms of α-synuclein adopt different conformations depending on environmental conditions. Advances in structural biology have accelerated fibril characterization. However, it remains unclear which conformations predominate in biological settings because current methods typically not only require isolating fibrils from their native environments, but they also do not provide insight about flexible regions.

Spatially resolved DNP-assisted NMR illuminates the conformational ensemble of α-synuclein in intact viable cells.

The protein α-syn adopts a wide variety of conformations including an intrinsically disordered monomeric form and an α-helical rich membrane-associated form that is thought to play an important role in cellular membrane processes. However, despite the high affinity of α-syn for membranes, evidence that the α-helical form of α-syn is adopted inside cells has thus far been indirect. In cell DNP-assisted solid state NMR on frozen samples has the potential to report directly on the entire conformational ensemble.

Conformational ensembles explain NMR spectra of frozen intrinsically disordered proteins.

Protein regions which are intrinsically disordered, exist as an ensemble of rapidly interconverting structures. Cooling proteins to cryogenic temperatures for dynamic nuclear polarization (DNP) magic angle spinning (MAS) NMR studies suspends most of the motions, resulting in peaks that are broad but not featureless. To demonstrate that detailed conformational restraints can be retrieved from the peak shapes of frozen proteins alone, we developed and used a simulation framework to assign peak features to conformers in the ensemble.

Mössbauer studies of the ferryl, ferrous and ferric states of dehaloperoxidase from A. ornata.

Dehaloperoxidase (DHP) is a multi-functional catalytic globin from the marine worm A. ornata, whose physiological functions include oxygen transport and oxidation of toxic substrates present in its habitat. In the Fe(III) state, DHPA has an isomer shift of 0.

Stability of the nitroxide biradical AMUPol in intact and lysed mammalian cells.

Dynamic Nuclear Polarization (DNP) enhanced solid state NMR increases experimental sensitivity, potentially enabling detection of biomolecules at their physiological concentrations. The sensitivity of DNP experiments is due to the transfer of polarization from electron spins of free radicals to the nuclear spins of interest. Here, we investigate the reduction of AMUPol in both lysed and intact HEK293 cells.

Tyrosyl radicals in dehaloperoxidase: how nature deals with evolving an oxygen-binding globin to a biologically relevant peroxidase.

Dehaloperoxidase (DHP) from Amphitrite ornata, having been shown to catalyze the hydrogen peroxide-dependent oxidation of trihalophenols to dihaloquinones, is the first oxygen binding globin that possesses a biologically relevant peroxidase activity. The catalytically competent species in DHP appears to be Compound ES, a reactive intermediate that contains both a ferryl heme and a tyrosyl radical. By simulating the EPR spectra of DHP activated by H2O2, Thompson et al.

The role of the distal histidine in H2O2 activation and heme protection in both peroxidase and globin functions.

The distal histidine mutations of dehaloperoxidase-hemoglobin A (DHP A) to aspartate (H55D) and asparagine (H55N) have been prepared to study the role played by the distal histidine in both activation and protection against oxidation by radicals in heme proteins. The H55D and H55N mutants of DHP A have ~6-fold and ~11-fold lower peroxidase activities than wild type enzyme toward the oxidation of 2,4,6-trichlorophenol (TCP) to yield 2,6-dichloroquinone (DCQ) in the presence of H(2)O(2). The origin of the lower rate constants may be the solvent-exposed conformations of distal D55 and N55, which would have the dual effect of destabilizing the binding of H(2)O(2) to the heme iron, and of removing the acid-base catalyst necessary for the heterolytic O-O bond cleavage of heme-bound H(2)O(2) (i.

Characterization of dehaloperoxidase compound ES and its reactivity with trihalophenols.

Dehaloperoxidase (DHP), the oxygen transport hemoglobin from the terebellid polychaete Amphitrite ornata, is the first globin identified to possess a biologically relevant peroxidase activity. DHP has been shown to oxidize trihalophenols to dihaloquinones in a dehalogenation reaction that uses hydrogen peroxide as a substrate. Herein, we demonstrate that the first detectable intermediate following the addition of hydrogen peroxide to ferric DHP contains both a ferryl heme and a tyrosyl radical, analogous to Compound ES of cytochrome c peroxidase.