Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are clinically distinctive diseases that feature a common neuropathological hallmark of aggregated α-synuclein. Little is known about how differences in α-synuclein aggregate structure affect disease phenotype. Here, we amplified α-synuclein aggregates from PD and MSA brain extracts and analyzed the conformational properties using fluorescent probes, NMR spectroscopy and electron paramagnetic resonance. We also generated and analyzed several in vitro α-synuclein polymorphs. We found that brain-derived α-synuclein fibrils were structurally different to all of the in vitro polymorphs analyzed. Importantly, there was a greater structural heterogeneity among α-synuclein fibrils from the PD brain compared to those from the MSA brain, possibly reflecting on the greater variability of disease phenotypes evident in PD. Our findings have significant ramifications for the use of non-brain-derived α-synuclein fibrils in PD and MSA studies, and raise important questions regarding the one disease-one strain hypothesis in the study of α-synucleinopathies.

In this study, the effector role of Pdx (putidaredoxin) on cytochrome P450cam conformation is refined by attaching two different spin labels, MTSL or BSL (bifunctional spin-label) onto the F or G helices and using DEER (double electron-electron resonance) to measure the distance between labels. Recent EPR and crystallographic studies have observed that oxidized Pdx induces substrate-bound P450cam to change from the closed to the open state. However, this change was not observed by DEER in the reduced Pdx complex with carbon-monoxide-bound P450cam (Fe(2+)CO). In addition, recent NMR studies have failed to observe a change in P450cam conformation upon binding Pdx. Hence, resolving these issues is important for a full understanding the effector role of Pdx. Here we show that oxidized Pdx induces camphor-bound P450cam to shift from the closed to the open conformation when labeled on either the F or G helices with MTSL. BSL at these sites can either narrow the distance distribution widths dramatically or alter the extent of the conformational change. In addition, we report DEER spectra on a mixed oxidation state containing oxidized Pdx and ferrous CO-bound P450cam, showing that P450cam remains closed. This indicates that CO binding to the heme prevents P450cam from opening, overriding the influence exerted by Pdx binding. Finally, we report the open form P450cam crystal structure with substrate bound, which suggests that crystal packing effects may prevent conformational conversion. Using multiple labeling approaches, DEER provides a unique perspective to resolve how the conformation of P450cam depends on Pdx and ligand states.

We report on radical polarization and optically-driven liquid DNP using nitroxide radicals functionalized by photoexcitable fullerene derivatives. Pulse laser excitation of the fullerene moiety leads to transient nitroxide radical polarization that is one order of magnitude larger than that at the Boltzmann equilibrium. The life time of the radical polarization increases with the size of the fullerene derivative and is correlated with the electronic spin-lattice relaxation time T. Overhauser NMR signal enhancements of toluene solvent protons were observed under steady-state illumination, which replaced microwave irradiation.

Cytochrome P450 CYP101A1 (P450cam) hydroxylates camphor by receiving two distinct electrons from its unique reductase, putidaredoxin (Pdx). Upon binding ferric P450cam, Pdx is now known to trigger a conformational change in the enzyme. This Pdx-induced conversion may provide the trigger to coordinate enzyme turnover and protect the enzyme from oxidative damage, so the interactions responsible for this conversion are of significant interest at present. This proposed role for Pdx requires that its interactions with P450cam be different for the open and closed conformations. In this study, we show that the binding thermodynamics of Pdx does indeed differ in the predicted way when the conformation of P450cam is held in different states. However, double electron-electron resonance measurements of intermolecular distances in the Pdx/P450cam complex show that the geometry of the complex is nearly identical for the open and closed states of P450cam. These studies show that Pdx appears to make a single distinct interaction with its binding site on the enzyme and triggers the conformational change through very subtle structural interactions.

The significant impact of the human virome on human physiology is beginning to emerge thanks to modern sequencing methods and bioinformatic tools. Anelloviruses, the principal constituent of the commensal human virome, are universally acquired in infancy and found throughout the body. Since the discovery of the original torque teno virus in 1997, three genera of the Anelloviridae family, each extremely diverse genetically, have been found in humans. These viruses elicit weak immune responses that permit multiple strains to co-exist and persist for years in a typical individual. However, because they do not cause disease and due to the lack of an in vitro culture system, anelloviruses remain poorly understood. Basic features of the virus, such as the identity of its structural protein, have been unclear until now. Here, we describe the first structure of an anellovirus particle, which includes a jelly roll domain that forms a 60-mer icosahedral particle core from which spike domains extend to form a salient part of the particle surface. The spike domains come together around the 5-fold symmetry axes to form crown-like features. Relatively conserved patches of amino acids are near the base of the spike domain while a hypervariable region is at the apex. We propose that this structure renders the particle less susceptible to antibody neutralization by hiding vulnerable conserved epitopes while exposing highly diverse epitopes as immunological decoys, thereby contributing to the immune evasion properties of anelloviruses. This would contrast with viruses such as beak and feather disease virus, canine parvovirus or adeno-associated virus which lack such pronounced surface features. These results shed light on the structure of anelloviruses and provide a framework to understand their interactions with the immune system.