UV Resonance Raman Structural Characterization of an (In)soluble Polyglutamine Peptide

Jakubek, Ryan S.; White, Stephen E.; Asher, Sanford A.

doi:10.1021/acs.jpcb.8b10783

Cited by 10 publications

(29 citation statements)

References 77 publications

(284 reference statements)

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“…Our recent study on polyglutamine-containing polypeptides showed that the organic solvents, TFA and HFIP aid in solubilisation by promoting the exchange of strong and intricate side chain-side chain and side chain-main chain hydrogen bonds present within the lyophilized peptides with themselves [30]. Similar observations were reported later by Jakubek et al (2019) as well based on UV resonance Raman spectroscopy analysis [40].…”

Section: Introductionsupporting

confidence: 79%

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

Burra

Maina

Serpell

et al. 2022

Preprint

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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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Section: Introductionsupporting

confidence: 79%

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

Burra

Maina

Serpell

et al. 2022

Preprint

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“…We compare our results with those previously published for Q10 and Q20 to investigate the dependence of the solution-state polyQ structures on repeat length. We find that disaggregated Q15 (DQ15) has predominately the same PPII-like structure as previously found for DQ10 and disaggregated Q20 (DQ20) . Also, non-disaggregated Q15 (NDQ15) has predominately the same collapsed β-strand-like conformation as previously found for NDQ10 .…”

Section: Introductionmentioning

confidence: 84%

“…UV resonance Raman (UVRR) spectroscopy has provided detailed insight into the secondary structures of polyQ peptides. − UVRR spectroscopy is a powerful tool for studying protein structure, solvation, and hydrogen bonding interactions. − Excitation in the deep UV (∼200 nm) selectively enhances vibrations of the secondary amide peptide backbone and primary amide glutamine (Gln) side chains. , These resonance-enhanced bands are sensitive to the structure and environment of the peptide backbone and Gln side chains …”

Section: Introductionmentioning

confidence: 99%

Polyglutamine Solution-State Structural Propensity Is Repeat Length Dependent

Jakubek¹,

Workman

White³

et al. 2019

J. Phys. Chem. B

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Expanded polyglutamine (polyQ) tracts in protein, which are known to induce their aggregation, are associated with numerous neurodegenerative diseases. Longer polyQ tracts correlate with faster protein aggregation kinetics and a decreased age of onset for polyQ disease symptoms. Here, we use UV resonance Raman spectroscopy, circular dichroism spectroscopy, and metadynamics simulations to investigate the solution-state structures of the D 2 Q 15 K 2 (Q15) and D 2 Q 20 K 2 (Q20) peptides. Using metadynamics, we explore the conformational energy landscapes of Q15 and Q20 and investigate the relative energies and activation barriers between these lowenergy structures. We compare the solution-state structures of D 2 Q 10 K 2 (Q10), Q15, and Q20 to determine the dependence of polyQ structure on the Q tract length. We show that these peptides can adopt two distinct monomeric conformations: an aggregation-resistant PPII-like conformation and an aggregation-prone β-strand-like conformation. We find that longer polyQ peptides have an increased preference for the aggregation-prone β-strand-like conformation. This preference may play an important role in the increased aggregation rate of longer polyQ peptides that are thought to lead to decreased neurodegenerative disease age of onset for polyQ disease patients.

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“…Lyophilized Httex1 proteins were disaggregated as described previously [85,[135][136][137] and resolubilized in 10 mM PBS, pH 7.2-7.4. Proteins were filtered prior to aggregation at 37 °C, and the soluble protein fraction was monitored over time by RP-UHPLC, as described previously [85,135].…”

Section: In Vitro Sedimentation Assaymentioning

confidence: 99%

The Nt17 domain and its helical conformation regulate the aggregation, cellular properties and neurotoxicity of mutant huntingtin exon 1

Vieweg

Mahul‐Mellier

Ruggeri

et al. 2021

Preprint

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The first 17 N-terminal amino acids (the Nt17 domain) flanking the polyQ tract of the Huntingtin protein (Htt) play an important role in modulating its aggregation, life cycle, membrane binding, and toxicity. Therefore, a better understanding of the molecular and structural determinants of the Nt17 code would likely provide important insights and help guide the development of future anti-aggregation and Htt lowering therapeutic strategies. Towards this goal, we sought to elucidate the role of the Nt17 sequence and helical conformation in regulating mutant Httex1 aggregation, morphology, uptake, and neuronal toxicity. To modulate the helical conformation of Nt17, we used a helix and membrane-binding disrupting mutation (M8P) strategy and site-specific introduction of post-translational modifications that are known to enhance (pT3) or disrupt (pS13, pS16, or pS13/pS16) the overall helicity of Nt17. Our in vitro studies show that the Nt17 and polyQ domains synergistically promote Httex1 aggregation, consistent with previous findings. However, we show that the Nt17 sequence, but not its helical conformation, is a key determinant of the morphology and growth of Httex1 fibrils. In cells, we show that the aggregation propensity and the toxic properties of de novo Httex1 were dependent on both the Nt17 sequence and its helical conformation and the synergistic effect of the Nt17 and polyQ domains. Finally, we demonstrate that the uptake of Httex1 into primary striatal neurons is strongly influenced by the helical propensity of Nt17. Phosphorylation (at T3 or S13/S16) or removal of the Nt17 domain increases the uptake and accumulation of Httex1 fibrils into the nucleus and induces neuronal cell death. Altogether our results demonstrate that the Nt17 domain serves as one of the key master regulators of Htt aggregation and toxicity and represents an attractive target for inhibiting Htt aggregate formation, inclusion formation, cell-to-cell propagation, and neuronal toxicity. These findings have significant implications for targeting the Nt17 domain to develop new disease-modifying therapies for the treatment of Huntington's disease.

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UV Resonance Raman Structural Characterization of an (In)soluble Polyglutamine Peptide

Cited by 10 publications

References 77 publications

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

GNNQQNY: Methodology for biophysical and structural understanding of aggregation

Polyglutamine Solution-State Structural Propensity Is Repeat Length Dependent

The Nt17 domain and its helical conformation regulate the aggregation, cellular properties and neurotoxicity of mutant huntingtin exon 1

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