GNNQQNY: Methodology for biophysical and structural understanding of aggregation

GNNQQNY sequence offers crucial information about the formation and structure of an amyloid fibril. In this study, we demonstrate a reproducible solubilisation protocol where the reduction of pH to 2.0 resulted in the generation of GNNQQNY monomers. The subsequent ultracentrifugation step removes the residual insoluble peptide from the homogeneous solution. This procedure ensures and allows the peptides to remain monomers till their aggregation is triggered by adjusting the pH to 7.2. The aggregation kinetics analysis showed a distinct lag-phase that is concentration-dependent, indicating nucleation-dependent aggregation kinetics. Nucleation kinetics analysis suggested a critical nucleus of size ~7 monomers at physiological conditions. The formed nucleus acts as a template for further self-assembly leading to the formation of highly ordered amyloid fibrils. These findings suggest that the proposed solubilisation protocol provides the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation in GNNQQNY sequences. This procedure can also be used for solubilising such small amyloidogenic sequences for their biophysical studies.

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Nucleation-dependent aggregation kinetics of Yeast Sup35 fragment GNNQQNY

10.1101/2020.07.27.221150 ◽

2020 ◽

Author(s):

Gunasekhar Burra ◽

Mahmoud B. Maina ◽

Louise C. Serpell ◽

Ashwani K. Thakur

Keyword(s):

Self Assembly ◽

Amyloid Fibrils ◽

Peptide Sequence ◽

Aggregation Kinetics ◽

Assembly Mechanism ◽

Structural Identity ◽

Different Temperatures ◽

Dynamics Simulations ◽

Amyloid Assembly ◽

Kinetics Of

AbstractAn N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY serves as an ideal model for structural understanding of amyloid assembly and kinetics. In this study, we used a reproducible solubilisation protocol that allows the generation of homogenous monomeric solution of GNNQQNY to understand the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequences follow nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the size and efficiency of nucleation was found to be inversely related to the reaction temperature. The generated nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T positivity, Congo red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers exerted no significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous predictions based on theoretical and molecular dynamics simulations. These findings will provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic systems associated with many systemic and neurodegenerative diseases.

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Kinetic Constraints in the Specific Interaction between Phosphorylated Ubiquitin and Proteasomal Shuttle Factors

Biomolecules ◽

10.3390/biom11071008 ◽

2021 ◽

Vol 11 (7) ◽

pp. 1008

Author(s):

Ling-Yun Qin ◽

Zhou Gong ◽

Kan Liu ◽

Xu Dong ◽

Chun Tang

Keyword(s):

Exchange Rate ◽

Self Assembly ◽

Solution Structure ◽

Specific Interaction ◽

Dissociation Rate ◽

Fine Tuning ◽

Binding Mechanism ◽

Kinetics Analysis ◽

Kinetic Constraint ◽

Thermodynamic Basis

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.

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Observation of an α-synuclein liquid droplet state and its maturation into Lewy body-like assemblies

Journal of Molecular Cell Biology ◽

10.1093/jmcb/mjaa075 ◽

2021 ◽

Author(s):

Maarten C Hardenberg ◽

Tessa Sinnige ◽

Sam Casford ◽

Samuel Dada ◽

Chetan Poudel ◽

...

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Self Assembly ◽

Lewy Body ◽

Amyloid Fibrils ◽

Liquid Droplet ◽

Large Body ◽

Lewy Bodies ◽

Cellular Components

Abstract Misfolded α-synuclein is a major component of Lewy bodies, which are a hallmark of Parkinson’s disease. A large body of evidence shows that α-synuclein can aggregate into amyloid fibrils, but the relationship between α-synuclein self-assembly and Lewy body formation remains unclear. Here we show, both in vitro and in a Caenorhabditis elegans model of Parkinson’s disease, that α-synuclein undergoes liquid‒liquid phase separation by forming a liquid droplet state, which converts into an amyloid-rich hydrogel with Lewy-body-like properties. This maturation process towards the amyloid state is delayed in the presence of model synaptic vesicles in vitro. Taken together, these results suggest that the formation of Lewy bodies may be linked to the arrested maturation of α-synuclein condensates in the presence of lipids and other cellular components.

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Examining the structure of the mature amyloid fibril

Biochemical Society Transactions ◽

10.1042/bst0300521 ◽

2002 ◽

Vol 30 (4) ◽

pp. 521-525 ◽

Cited By ~ 43

Author(s):

O. S. Makin ◽

L. C. Serpell

Keyword(s):

Self Assembly ◽

Rational Design ◽

Amyloid Fibrils ◽

Structural Information ◽

X Ray ◽

X Ray Crystallography ◽

Cryo Electron Microscopy ◽

Fibre Diffraction ◽

Complementary Techniques ◽

Mature Fibril

The pathogenesis of the group of diseases known collectively as the amyloidoses is characterized by the deposition of insoluble amyloid fibrils. These are straight, unbranching structures about 70–120 å (1 å = 0.1 nm) in diameter and of indeterminate length formed by the self-assembly of a diverse group of normally soluble proteins. Knowledge of the structure of these fibrils is necessary for the understanding of their abnormal assembly and deposition, possibly leading to the rational design of therapeutic agents for their prevention or disaggregation. Structural elucidation is impeded by fibril insolubility and inability to crystallize, thus preventing the use of X-ray crystallography and solution NMR. CD, Fourier-transform infrared spectroscopy and light scattering have been used in the study of the mechanism of fibril formation. This review concentrates on the structural information about the final, mature fibril and in particular the complementary techniques of cryo-electron microscopy, solid-state NMR and X-ray fibre diffraction.

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Mobilization of Selenite by Ralstonia metallidurans CH34

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.769-773.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 769-773 ◽

Cited By ~ 73

Author(s):

Murielle Roux ◽

Géraldine Sarret ◽

Isabelle Pignot-Paintrand ◽

Marc Fontecave ◽

Jacques Coves

Keyword(s):

Alcaligenes Eutrophus ◽

Culture Conditions ◽

Lag Phase ◽

Elemental Selenium ◽

Kinetics Analysis ◽

X Ray ◽

Polluted Sites ◽

Ralstonia Metallidurans ◽

X Ray Absorption ◽

Bioremediation Processes

ABSTRACT Ralstonia metallidurans CH34 (formerlyAlcaligenes eutrophus CH34) is a soil bacterium characteristic of metal-contaminated biotopes, as it is able to grow in the presence of a variety of heavy metals. R. metalliduransCH34 is reported now to resist up to 6 mM selenite and to reduce selenite to elemental red selenium as shown by extended X-ray absorption fine-structure analysis. Growth kinetics analysis suggests an adaptation of the cells to the selenite stress during the lag-phase period. Depending on the culture conditions, the medium can be completely depleted of selenite. Selenium accumulates essentially in the cytoplasm as judged from electron microscopy and energy-dispersive X-ray analysis. Elemental selenium, highly insoluble, represents a nontoxic storage form for the bacterium. The ability of R. metallidurans CH34 to reduce large amounts of selenite may be of interest for bioremediation processes targeting selenite-polluted sites.

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ChemInform Abstract: Self-Assembly of Polypeptides into Amyloid Fibrils with Structural Transitions.

ChemInform ◽

10.1002/chin.200224288 ◽

2010 ◽

Vol 33 (24) ◽

pp. no-no

Author(s):

Hisakazu Mihara ◽

Yuta Takahashi

Keyword(s):

Self Assembly ◽

Amyloid Fibrils ◽

Structural Transitions

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RETRACTED: Peptide-induced formation of protein aggregates and amyloid fibrils in human and guinea pig αA-crystallins under physiological conditions of temperature and pH

Experimental Eye Research ◽

10.1016/j.exer.2018.11.016 ◽

2019 ◽

Vol 179 ◽

pp. 193-205 ◽

Cited By ~ 1

Author(s):

Anbarasu Kumarasamy ◽

Sivakumar Jeyarajan ◽

Jonathan Cheon ◽

Anthony Premceski ◽

Eric Seidel ◽

...

Keyword(s):

Guinea Pig ◽

Amyloid Fibrils ◽

Protein Aggregates ◽

Physiological Conditions

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Quantification of amyloid fibril polymorphism by nano-morphometry reveals the individuality of filament assembly

Communications Chemistry ◽

10.1038/s42004-020-00372-3 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Liam D. Aubrey ◽

Ben J. F. Blakeman ◽

Liisa Lutter ◽

Christopher J. Serpell ◽

Mick F. Tuite ◽

...

Keyword(s):

Self Assembly ◽

Amyloid Fibrils ◽

Amyloid Fibril ◽

Biological Function ◽

Structural Basis ◽

Distance Curve ◽

Filament Assembly ◽

Sample Heterogeneity ◽

Structural Insights ◽

Polymorphic Nature

Abstract Amyloid fibrils are highly polymorphic structures formed by many different proteins. They provide biological function but also abnormally accumulate in numerous human diseases. The physicochemical principles of amyloid polymorphism are not understood due to lack of structural insights at the single-fibril level. To identify and classify different fibril polymorphs and to quantify the level of heterogeneity is essential to decipher the precise links between amyloid structures and their functional and disease associated properties such as toxicity, strains, propagation and spreading. Employing gentle, force-distance curve-based AFM, we produce detailed images, from which the 3D reconstruction of individual filaments in heterogeneous amyloid samples is achieved. Distinctive fibril polymorphs are then classified by hierarchical clustering, and sample heterogeneity is objectively quantified. These data demonstrate the polymorphic nature of fibril populations, provide important information regarding the energy landscape of amyloid self-assembly, and offer quantitative insights into the structural basis of polymorphism in amyloid populations.

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