Exploration of Hinge Residues among GFP-like Proteins

Before the maturation of the chromophore, a fluorescent protein, just like any other three-dimensional protein structure, has to fold into the correct tertiary structure to function. We proposed that the evolutionarily conserved hinge residues, believed to be located on or near the lids of the fluorescent protein, are involved in the folding mechanism and rotation of the β-sheets and lids into the correct geometry. Acting like door hinges, these residues are translationally immobilized but rotationally active. Interference of the hinge sites may lead to allosteric effects and disruption of the protein’s functional motions. In the study, significant sequential and spatial conservation was found in conserved lid residues across 28 wild-type fluorescent proteins. Furthermore, the high rotational freedom and dihedral mobility of the conserved lid residues confirmed their behavior as hinge residues in the folding process

Water diffusion in and out of the β-barrel of GFP and the fast maturing fluorescent protein, TurboGFP

The chromophore of fluorescent proteins is formed by an internal cyclization of the tripeptide 65SYG67 fragment and a subsequent oxidation. The oxidation is slow – the kinetics of this step is presumably improved in fast maturing GFPs. Water molecules can aid in the chromophore formation. We have used 50ns molecular dynamics simulations of the mature and immature forms of avGFP and TurboGFP to examine the diffusion of water molecules in-and-out of the protein β-barrel. Most crystal structures of GFPs have well-structured waters within hydrogen-bonding distance of Glu222 and Arg96. It has been proposed that they have an important role in chromophore formation. Stable waters are found in similar positions in all simulations conducted. The simulations confirm the existence of a pore that leads to the chromophore in the rapidly maturing TurboGFP; decreased water diffusion upon chromophore formation; and increased water diffusion due to the pore formation

Different Amyloid-β Self-Assemblies Have Distinct Effects on Intracellular Tau Aggregation.

Alzheimer's disease (AD) pathology is characterized by the aggregation of beta-amyloid (Aβ) and tau in the form of amyloid plaques and neurofibrillary tangles in the brain. It has been found that a synergistic relationship between these two proteins may contribute to their roles in disease progression. However, how Aβ and tau interact has not been fully characterized. Here, we analyze how tau seeding or aggregation is influenced by different Aβ self-assemblies (fibrils and oligomers). Our cellular assays utilizing tau biosensor cells show that transduction of Aβ oligomers into the cells greatly enhances seeded tau aggregation in a concentration-dependent manner. In contrast, transduced Aβ fibrils slightly reduce tau seeding while untransduced Aβ fibrils promote it. We also observe that the transduction of α-synuclein fibrils, another amyloid protein, has no effect on tau seeding. The enhancement of tau seeding by Aβ oligomers was confirmed using tau fibril seeds derived from both recombinant tau and PS19 mouse brain extracts containing human tau. Our findings highlight the importance of considering the specific form and cellular location of Aβ self-assembly when studying the relationship between Aβ and tau in future AD therapeutic development

Structures of fibrils formed by α-synuclein hereditary disease mutant H50Q reveal new polymorphs.

Deposits of amyloid fibrils of α-synuclein are the histological hallmarks of Parkinson's disease, dementia with Lewy bodies and multiple system atrophy, with hereditary mutations in α-synuclein linked to the first two of these conditions. Seeing the changes to the structures of amyloid fibrils bearing these mutations may help to understand these diseases. To this end, we determined the cryo-EM structures of α-synuclein fibrils containing the H50Q hereditary mutation. We find that the H50Q mutation results in two previously unobserved polymorphs of α-synuclein: narrow and wide fibrils, formed from either one or two protofilaments, respectively. These structures recapitulate conserved features of the wild-type fold but reveal new structural elements, including a previously unobserved hydrogen-bond network and surprising new protofilament arrangements. The structures of the H50Q polymorphs help to rationalize the faster aggregation kinetics, higher seeding capacity in biosensor cells and greater cytotoxicity that we observe for H50Q compared to wild-type α-synuclein

Cryo-EM of full-length α-synuclein reveals fibril polymorphs with a common structural kernel.

α-Synuclein (aSyn) fibrillar polymorphs have distinct in vitro and in vivo seeding activities, contributing differently to synucleinopathies. Despite numerous prior attempts, how polymorphic aSyn fibrils differ in atomic structure remains elusive. Here, we present fibril polymorphs from the full-length recombinant human aSyn and their seeding capacity and cytotoxicity in vitro. By cryo-electron microscopy helical reconstruction, we determine the structures of the two predominant species, a rod and a twister, both at 3.7 Å resolution. Our atomic models reveal that both polymorphs share a kernel structure of a bent β-arch, but differ in their inter-protofilament interfaces. Thus, different packing of the same kernel structure gives rise to distinct fibril polymorphs. Analyses of disease-related familial mutations suggest their potential contribution to the pathogenesis of synucleinopathies by altering population distribution of the fibril polymorphs. Drug design targeting amyloid fibrils in neurodegenerative diseases should consider the formation and distribution of concurrent fibril polymorphs

Cryo-EM Structures and Biological Study of Pathogenic Alpha-Synuclein Fibrils

Multiple neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) have been associated with the pathological aggregation of amyloid protein α-synuclein (αSyn). Similar to tau strains from different tauopathies, strains of αSyn have been demonstrated distinct cell-to-cell spreading. The distinct biological activity from each strain is encoded by the specific conserved conformation. However, we knew little about the atomic structures of αSyn fibril polymorphs. Our works revealed cryo-EM structures of αSyn wild-type fibril polymorphs and αSyn fibril polymorphs with hereditary mutation (E46K and H50Q). We showed two types of kernel that can be found in all αSyn polymorphs, which is validated by the fibrils from MSA patients. We also found αSyn fibril polymorphs are differed by varying 1) folding of protofilament 2) number of protofilament 3) binding interface between protofilaments, thus leads to distinct cellular toxicity, aggregation rate and seeding capacity. Furthermore, we extracted and characterized fibrils from patients with Parkinson’s disease, which formed a different polymorph. The study provides near-atomic insights about αSyn aggregation and enables future development of therapeutics

Cryo-EM Structures and Biological Study of Pathogenic Alpha-Synuclein Fibrils

Multiple neurodegenerative diseases, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) have been associated with the pathological aggregation of amyloid protein α-synuclein (αSyn). Similar to tau strains from different tauopathies, strains of αSyn have been demonstrated distinct cell-to-cell spreading. The distinct biological activity from each strain is encoded by the specific conserved conformation. However, we knew little about the atomic structures of αSyn fibril polymorphs. Our works revealed cryo-EM structures of αSyn wild-type fibril polymorphs and αSyn fibril polymorphs with hereditary mutation (E46K and H50Q). We showed two types of kernel that can be found in all αSyn polymorphs, which is validated by the fibrils from MSA patients. We also found αSyn fibril polymorphs are differed by varying 1) folding of protofilament 2) number of protofilament 3) binding interface between protofilaments, thus leads to distinct cellular toxicity, aggregation rate and seeding capacity. Furthermore, we extracted and characterized fibrils from patients with Parkinson’s disease, which formed a different polymorph. The study provides near-atomic insights about αSyn aggregation and enables future development of therapeutics

Plane Machining by Inner-Jet Electrochemical Milling of TiB2/7050 Aluminum Matrix Composite

Electrochemical milling (ECM) is an ideal technique for machining thin-walled structural parts of aluminum matrix composites. Adopting a reasonable tool cathode structure, feed rate, and processing method can improve the machining efficiency. In this study, a tool cathode with a reasonable structure was selected through flow field simulation. Then, the material removal rate (MRR) and surface roughness were studied using various ECM parameters. Finally, the transverse movement and processing method in which the starting position was rotated 90° were studied, and a plane of 59 × 59 mm was machined. The experimental results show that using an appropriate tool cathode can create a more uniform flow field. The MRR was 168.6 mm3/min and the surface roughness (Ra) was 3.329 µm at a feed rate of 30 mm/min. For machining larger plane structures, a transverse movement of 7 mm is verified to be the most suitable because of the best smoothness in the middle of the two processes. By using the same machining method and rotating the starting position 90°, the flatness of the processing plane decreased from 0.296 mm to 0.251 mm, a reduction of 15.2% compared to that obtained in the first processing