Papers by Anthony William Paul Fitzpatrick
Cell, 2022
Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentou... more Misfolding and aggregation of disease-specific proteins, resulting in the formation of filamentous cellular inclusions, is a hallmark of neurodegenerative disease with characteristic filament structures, or conformers, defining each proteinopathy. Here we show that a previously unsolved amyloid fibril composed of a 135 amino acid C-terminal fragment of TMEM106B is a common finding in distinct human neurodegenerative diseases, including cases characterized by abnormal aggregation of TDP-43, tau, or α-synuclein protein. A combination of cryoelectron microscopy and mass spectrometry was used to solve the structures of TMEM106B fibrils at a resolution of 2.7 Å from postmortem human brain tissue afflicted with frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP, n = 8), progressive supranuclear palsy (PSP, n = 2), or dementia with Lewy bodies (DLB, n = 1). The commonality of abundant amyloid fibrils composed of TMEM106B, a lysosomal/endosomal protein, to a broad range of debilitating human disorders indicates a shared fibrillization pathway that may initiate or accelerate neurodegeneration.
Chang et al., 2022
Highlights: Cryo-EM structures of brain-derived TMEM106B fibrils from neurodegenerative diseases ... more Highlights: Cryo-EM structures of brain-derived TMEM106B fibrils from neurodegenerative diseases d Endolysosomal membrane protein TMEM106B C-terminal fragment forms amyloid fibrils. TMEM106B fibrillization is widespread among diverse neurodegenerative proteinopathies. Identification of fibrillization pathway potentially implicated in neurodegeneration.
Misfolding and aggregation of tau protein is implicated in many neurodegenerative diseases that a... more Misfolding and aggregation of tau protein is implicated in many neurodegenerative diseases that are typified by the presence of large, filamentous tau inclusions. The aggregation of tau in human brain is disease-specific with characteristic filaments defining the neuropathology. An understanding of how identical tau isoforms aggregate into disparate filament morphologies in phenotypically distinct tau-related diseases remains elusive. Here, we determine the structure of a brain-derived twisted tau filament in progressive supranuclear palsy and compare it to a dissimilar tau fold found in corticobasal degeneration. While the tau filament core in both diseases is comprised of residues 274 to 380, molecular-level polymorphism exists. Potential origins of the molecular polymorphism, such as noncovalent cofactor binding, are identified and predicted to modulate tau filament structures in the brain.
Proceedings of the National Academy of Sciences of the United States of America, Jan 2, 2015
The amyloid state of polypeptides is a stable, highly organized structural form consisting of lat... more The amyloid state of polypeptides is a stable, highly organized structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly anisotropic bioma...
PLoS ONE, 2013
a-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson's disease and... more a-Synuclein is a small protein strongly implicated in the pathogenesis of Parkinson's disease and related neurodegenerative disorders. We report here the use of in-cell NMR spectroscopy to observe directly the structure and dynamics of this protein within E. coli cells. To improve the accuracy in the measurement of backbone chemical shifts within crowded in-cell NMR spectra, we have developed a deconvolution method to reduce inhomogeneous line broadening within cellular samples. The resulting chemical shift values were then used to evaluate the distribution of secondary structure populations which, in the absence of stable tertiary contacts, are a most effective way to describe the conformational fluctuations of disordered proteins. The results indicate that, at least within the bacterial cytosol, a-synuclein populates a highly dynamic state that, despite the highly crowded environment, has the same characteristics as the disordered monomeric form observed in aqueous solution.
The Journal of Physical Chemistry B, 2010
Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated wit... more Amyloid fibrils are structurally ordered aggregates of proteins whose formation is associated with many neurodegenerative and other diseases. For that reason, their high-resolution structures are of considerable interest and have been studied using a wide range of techniques, notably electron microscopy, X-ray diffraction, and magic angle spinning (MAS) NMR. Because of the excellent resolution in the spectra, MAS NMR is uniquely capable of delivering site-specific, atomic resolution information about all levels of amyloid structure: (1) the monomer, which packs into several (2) protofilaments that in turn associate to form a (3) fibril. Building upon our high-resolution structure of the monomer of an amyloid-forming peptide from transthyretin (TTR 105-115), we introduce single 1-13 C labeled amino acids at seven different sites in the peptide and measure intermolecular carbonyl-carbonyl distances with an accuracy of 0.11 A. Our results conclusively establish a parallel, in register, topology for the packing of this peptide into a-sheet and provide constraints essential for the determination of an atomic resolution structure of the fibril. Furthermore, the approach we employ, based on a combination of a double-quantum filtered variant of the DRAWS recoupling sequence and multispin numerical simulations in SPINEVOLUTION, is general and should be applicable to a wide range of systems.
The Journal of Physical Chemistry B, 2008
We employ constant-temperature and replica exchange molecular dynamics to survey the free energy ... more We employ constant-temperature and replica exchange molecular dynamics to survey the free energy landscape of the cc peptide using a united-atom potential and an implicit solvent representation. Starting from the experimental coiled-coil structure we observe R to conversion on increasing the temperature, in agreement with experiment. Various-sheet trimers are identified as free energy minima, including one that closely resembles the amyloid-sheet model previously proposed from experimental data. We characterize two alternative pathways leading to-sheets. The first proceeds via direct R to conversion without dissociation of the trimer, and the second can be classified as a dissociation/reassociation pathway.
Science, 2007
Protein molecules have the ability to form a rich variety of natural and artificial structures an... more Protein molecules have the ability to form a rich variety of natural and artificial structures and materials. We show that amyloid fibrils, ordered supramolecular nanostructures that are self-assembled from a wide range of polypeptide molecules, have rigidities varying over four orders of magnitude, and constitute a class of high-performance biomaterials. We elucidate the molecular origin of fibril material properties and show that the major contribution to their rigidity stems from a generic interbackbone hydrogen-bonding network that is modulated by variable side-chain interactions.
Proceedings of the National Academy of Sciences, 2013
Amyloid is an important class of proteinaceous material because of its close association with pro... more Amyloid is an important class of proteinaceous material because of its close association with protein misfolding disorders such as Alzheimer’s disease and type II diabetes. Although the degree of stiffness of amyloid is critical to the understanding of its pathological and biological functions, current estimates of the rigidity of these β-sheet–rich protein aggregates range from soft (10 8 Pa) to hard (10 10 Pa) depending on the method used. Here, we use time-resolved 4D EM to directly and noninvasively measure the oscillatory dynamics of freestanding, self-supporting amyloid beams and their rigidity. The dynamics of a single structure, not an ensemble, were visualized in space and time by imaging in the microscope an amyloid–dye cocrystal that, upon excitation, converts light into mechanical work. From the oscillatory motion, together with tomographic reconstructions of three studied amyloid beams, we determined the Young modulus of these highly ordered, hydrogen-bonded β-sheet str...
Proceedings of the National Academy of Sciences, 2013
The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible ... more The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structur...
PLoS Computational Biology, 2011
Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into the... more Identifying the forces that drive proteins to misfold and aggregate, rather than to fold into their functional states, is fundamental to our understanding of living systems and to our ability to combat protein deposition disorders such as Alzheimer's disease and the spongiform encephalopathies. We report here the finding that the balance between hydrophobic and hydrogen bonding interactions is different for proteins in the processes of folding to their native states and misfolding to the alternative amyloid structures. We find that the minima of the protein free energy landscape for folding and misfolding tend to be respectively dominated by hydrophobic and by hydrogen bonding interactions. These results characterise the nature of the interactions that determine the competition between folding and misfolding of proteins by revealing that the stability of native proteins is primarily determined by hydrophobic interactions between sidechains, while the stability of amyloid fibrils depends more on backbone intermolecular hydrogen bonding interactions.
Physical Review Letters, 2012
We study two distinctly ordered condensed phases of polypeptide molecules, amyloid fibrils and am... more We study two distinctly ordered condensed phases of polypeptide molecules, amyloid fibrils and amyloidlike microcrystals, and the first-order twisting phase transition between these two states. We derive a single free-energy form which connects both phases. Our model identifies relevant degrees of freedom for describing the collective behavior of supramolecular polypeptide structures, reproduces accurately the results from molecular dynamics simulations as well as from experiments, and sheds light on the uniform nature of the dimensions of different peptide fibrils.
Journal of the American Chemical Society, 2011
An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils rev... more An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.
Journal of the American Chemical Society, 2013
Cryo-electron microscopy is a form of transmission electron microscopy that has been used to dete... more Cryo-electron microscopy is a form of transmission electron microscopy that has been used to determine the 3D structure of biological specimens in the hydrated state and with high resolution. We report the development of 4D cryo-electron microscopy by integrating the fourth dimension, time, into this powerful technique. From time-resolved diffraction of amyloid fibrils in a thin layer of vitrified water at cryogenic temperatures, we were able to detect picometer movements of protein molecules on a nanosecond time scale. Potential future applications of 4D cryo-electron microscopy are numerous, and some are discussed here.
Journal of the American Chemical Society, 2013
Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several am... more Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure, and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105−115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide β-strands into β-sheets but also the β-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The β-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N-and C-termini of the peptide and in inhomogeneous line-broadening for the residues buried within the interior of the fibrils.
Current Opinion in Structural Biology
Neurodegenerative and other protein misfolding diseases are associated with the aggregation of a ... more Neurodegenerative and other protein misfolding diseases are associated with the aggregation of a protein, which may be mutated in genetic forms of disease, or the wild type form in late onset sporadic disease. A wide variety of proteins and peptides can be involved, with aggregation originating from a natively folded or a natively unstructured species. Large deposits of amyloid fibrils are typically associated with cell death in late stage pathology. In this review, we illustrate the contributions of cryo-EM and related methods to the structure determination of amyloid fibrils extracted post mortem from patient brains or formed in vitro. We also discuss cell models of protein aggregation and the contributions of electron tomography to understanding the cellular context of aggregation.
Cell
Highlights d Cryo-EM structures of human brain-derived tau filaments from a four-repeat tauopathy... more Highlights d Cryo-EM structures of human brain-derived tau filaments from a four-repeat tauopathy d Cryo-EM and MS of tau filaments enable PTM mapping directly onto atomic models d Comparing tauopathies reveals that ubiquitination of tau can mediate fibril diversity d Integrated structural biology elucidates principles of PTMmediated fibril assembly
The amyloid state of polypeptides is a stable, highly organized
structural form consisting of lat... more The amyloid state of polypeptides is a stable, highly organized
structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly
anisotropic biomaterial in vivo is discussed.
Cryo-electron microscopy is a form of transmission electron microscopy that has been used to dete... more Cryo-electron microscopy is a form of transmission electron microscopy that has been used to determine the 3D structure of biological specimens in the hydrated state and with high resolution. We report the development of 4D cryo-electron microscopy by integrating the fourth dimension, time, into this powerful technique. From time-resolved diffraction of amyloid fibrils in a thin layer of vitrified water at cryogenic temperatures, we were able to detect picometer movements of protein molecules on a nanosecond time scale. Potential future applications of 4D cryo-electron microscopy are numerous, and some are discussed here. Communication pubs.acs.org/JACS
wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submi... more wrote the paper. The authors declare no conflict of interest. This article is a PNAS Direct Submission. Freely available online through the PNAS open access option. Data deposition: The structures of the TTR(105-115) amyloid fibrils have been deposited in the Protein Data Bank, www.pdb.org [PDB ID code 2m5n (Protofilament), 2m5k (Doublet), 2m5m (Triplet), and 3zpk (Quadruplet)] and the Electron Microscopy Data Bank [EMDB accession no. EMD-5590 (Doublet), EMD-2323 (Triplet), and EMD-2324 (Quadruplet)].
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Papers by Anthony William Paul Fitzpatrick
structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly
anisotropic biomaterial in vivo is discussed.
structural form consisting of laterally associated β-sheet protofilaments that may be adopted as an alternative to the functional, native state. Identifying the balance of forces stabilizing amyloid is fundamental to understanding the wide accessibility of this state to peptides and proteins with unrelated primary sequences, various chain lengths, and widely differing native structures. Here, we use four-dimensional electron microscopy to demonstrate that the forces acting to stabilize amyloid at the atomic level are highly anisotropic, that an optimized interbackbone hydrogen-bonding network within β-sheets confers 20 times more rigidity on the structure than sequence-specific sidechain interactions between sheets, and that electrostatic attraction of protofilaments is only slightly stronger than these weak amphiphilic interactions. The potential biological relevance of the deposition of such a highly
anisotropic biomaterial in vivo is discussed.