Aggregate Polymorphism in Protein Deposition Diseases: Investigations by Magic Angle Spinning Solid State NMR and Transmission Electron Microscopy

The deposition of normally soluble protein can occur in any organ in the human body and is associated with tissue dysfunction, cell death, and the progression of disease. Protein aggregation is concomitant with blindness as an outcome of cataract, life-threatening organ failure as a consequence of amyloidosis, and pronounced degeneration of the brain. The mutation responsible for Huntington’s disease causes an expansion of the polyglutamine domain of huntingtin exon 1 that directly promotes misfolding and refolding of huntingtin and huntingtin N-terminal fragments into amyloid-like fibrils in the basal striatum and cortex of the brain. Several fibril polymorphs have been identified, however the relationship between neurotoxicity and amyloid polymorphism is poorly understood. The P23T mutant of gamma-D-crystallin is associated with cataract formation in the eyes of very young children. Crystallins have been shown to form amyloid-like, native-like, as well as amorphous looking aggregates in vitro, accordingly it is unclear which class of aggregates P23T gamma-D-crystallin is most likely to form in cataract. Apolipoprotein A-I is a known anti-atherosclerotic factor and oxidation at methionine residues enhances its function. However, this oxidation also induces aggregation in vascular amyloidosis, which is interlinked with atherosclerosis progression. It is unclear whether apolipoprotein A-I aggregates misfold into amyloid-like fibrils as is usually the case in amyloidosis. Magic angle spinning solid state NMR (MAS ssNMR) is ideally suited to provide atomic resolution information on the structure and dynamics of insoluble, non-crystalline protein aggregates. Transmission electron microscopy (TEM) allows for the visualization of morphological features of aggregates that cannot be observed by optical microscopy and can be used to identify polymorphs and aid in distinguishing between different classes of aggregates. In this dissertation, I use both MAS ssNMR and TEM in addition to other biophysical and structural techniques to investigate the differences in structure and dynamics between polymorphs of huntingtin exon 1, P23T gamma-D-crystallin, and apolipoprotein A-I. Enabled by my experiments, I narrow down the potential molecular mechanisms involved in these three distinct types of protein deposition diseases. I show that depending on the milieu, proteins have the potential for varied amyloidogenic and non-amyloidogenic self-assembly

Selective observation of semi-rigid non-core residues in dynamically complex mutant huntingtin protein fibrils

Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein's expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils' segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of 'intermediate motion selection' (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter's detection with specific dyes and positron emission tomography tracers

Protofilament structure and supramolecular polymorphism of aggregated mutant huntingtin exon 1

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity

Huntingtin exon 1 fibrils feature an interdigitated β-hairpin-based polyglutamine core

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s Disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intra- and inter-molecular contacts, backbone and side chain torsion angles, relaxation measurements, and calculations of chemical shifts. These reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are co-assembled from differently structured monomers, which we describe as a type of ‘intrinsic’ polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. Weshow that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain, and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms

NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission

Peptide-Directed Assembly of Single-Helical Gold Nanoparticle Superstructures Exhibiting Intense Chiroptical Activity

Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C₁₈-(PEP_Au^M‑ox)₂ (PEP_Au^M‑ox = AYSSGA­PPM^oxPPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute <i>g</i>-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles. The morphology and assembled structure of C₁₈-(PEP_Au^M‑ox)₂ are studied using TEM, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, X-ray diffraction (XRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. TEM and AFM reveal that C₁₈-(PEP_Au^M‑ox)₂ assembles into linear amyloid-like 1D helical ribbons having structural parameters that correlate to those of the single-helical gold nanoparticle superstructures. FTIR, CD, XRD, and ssNMR indicate the presence of cross-β and polyproline II secondary structures. A molecular assembly model is presented that takes into account all experimental observations and that supports the single-helical nanoparticle assembly architecture. This model provides the basis for the design of future nanoparticle assemblies having programmable structures and properties