NMR determination of p<i>K</i><sub>a</sub> values in α‐synuclein

Croke, Robyn L.; Patil, Sharadrao M.; Quevreaux, Jason; Kendall, Debra A.; Alexandrescu, Andrei T.

doi:10.1002/pro.556

Cited by 61 publications

(96 citation statements)

References 70 publications

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“…C, NMR pH titration of histidine H⑀1 protons. The unresolved resonances from the His 6 tag used for purification gave a pK a of 6.7, in agreement with the random coil histidine value (43). By contrast His-305 had a pK a value shifted up by 2.5 pH units, consistent with the residue forming a stabilizing salt bridge with Asp-302.…”

Section: Resultssupporting

confidence: 68%

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Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Harprecht

Okifo

Robbins

et al. 2016

Journal of Biological Chemistry

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The I-domain is a genetic insertion in the phage P22 coat protein that chaperones its folding and stability. Of 11 acidic residues in the I-domain, seven participate in stabilizing electrostatic interactions with basic residues across elements of secondary structure, fastening the ␤-barrel fold. A hydrogenbonded salt bridge between Asp-302 and His-305 is particularly interesting as Asp-302 is the site of a temperature-sensitivefolding mutation. The pK a of His-305 is raised to 9.0, indicating the salt bridge stabilizes the I-domain by ϳ4 kcal/mol. Consistently, urea denaturation experiments indicate the stability of the WT I-domain decreases by 4 kcal/mol between neutral and basic pH. The mutants D302A and H305A remove the pH dependence of stability. The D302A substitution destabilizes the I-domain by 4 kcal/mol, whereas H305A had smaller effects, on the order of 1-2 kcal/mol. The destabilizing effects of D302A are perpetuated in the full-length coat protein as shown by a higher sensitivity to protease digestion, decreased procapsid assembly rates, and impaired phage production in vivo. By contrast, the mutants have only minor effects on capsid expansion or stability in vitro. The effects of the Asp-302-His-305 salt bridge are thus complex and context-dependent. Substitutions that abolish the salt bridge destabilize coat protein monomers and impair capsid self-assembly, but once capsids are formed the effects of the substitutions are overcome by new quaternary interactions between subunits.

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Section: Resultssupporting

confidence: 68%

“…where pK a is the logarithmic acid ionization constant, ␦ low is the low pH chemical shift plateau, ␦ high is the high pH chemical shift plateau, and n is the apparent Hill coefficient (42,43). Circular Dichroism (CD)-CD experiments were performed on a Pi-Star 180 spectropolarimeter from Applied Photophysics (Leatherhead, Surrey, UK).…”

Section: Methodsmentioning

confidence: 99%

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Harprecht

Okifo

Robbins

et al. 2016

Journal of Biological Chemistry

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“…Whereas the single histidine residue (H50) has a pK a value of 6.8, the ca. 4.4 units calculated change in net charge between pH 6.0 and 5.0 is mainly due to the partial protonation of several carboxylate groups in the protein, especially in the acidic C terminus, which contains 16 carboxylate groups, 13 of which have elevated pK a values (39). At lower protein concentrations and buffer strengths, as in the present study, as well as in fibrils with many negative groups close together, these pK a values are likely to be increased even more, in line with the salt dependence of α-synuclein pK a values (39) and similar studies (40).…”

Section: Discussionmentioning

confidence: 99%

“…SI Appendix, Fig. S12 shows the net charge of the protein, calculated as a function of pH, based on the pK a values and Hill coefficients reported for 250 μM α-syn in 20 mM PB (39). Whereas the single histidine residue (H50) has a pK a value of 6.8, the ca.…”

Section: Discussionmentioning

confidence: 99%

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

Buell

Galvagnion

Gaspar³

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

603

867

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The formation of amyloid fibrils by the intrinsically disordered protein α-synuclein is a hallmark of Parkinson disease. To characterize the microscopic steps in the mechanism of aggregation of this protein we have used in vitro aggregation assays in the presence of preformed seed fibrils to determine the molecular rate constant of fibril elongation under a range of different conditions. We show that α-synuclein amyloid fibrils grow by monomer and not oligomer addition and are subject to higher-order assembly processes that decrease their capacity to grow. We also find that at neutral pH under quiescent conditions homogeneous primary nucleation and secondary processes, such as fragmentation and surface-assisted nucleation, which can lead to proliferation of the total number of aggregates, are undetectable. At pH values below 6, however, the rate of secondary nucleation increases dramatically, leading to a completely different balance between the nucleation and growth of aggregates. Thus, at mildly acidic pH values, such as those, for example, that are present in some intracellular locations, including endosomes and lysosomes, multiplication of aggregates is much faster than at normal physiological pH values, largely as a consequence of much more rapid secondary nucleation. These findings provide new insights into possible mechanisms of α-synuclein aggregation and aggregate spreading in the context of Parkinson disease.seeding | prion-like behavior | neurodegenerative disease | kinetic analysis | electrostatic interactions T he conversion of soluble peptide and protein molecules into insoluble amyloid fibrils is of great interest in fields of science ranging from molecular medicine to nanotechnology (1). The formation of amyloid fibrils is a characteristic feature of a substantial number of increasingly common medical disorders, including neurodegenerative conditions such as Alzheimer's and Parkinson diseases (2). Elucidating the fundamental mechanistic steps involved in the conversion from the soluble to the fibrillar forms of the peptides and proteins involved in such disorders is crucial for understanding their origin and proliferation, and hence for exploring in a rational manner new and effective therapeutic strategies through which to combat their onset or progression (3).One general aspect of amyloid diseases is that once the first aggregates are formed it is very difficult to stop or reverse the aggregation process. This implies that aggregation needs to be studied in both the absence and presence of preformed aggregates, commonly known as seeds, to deepen our understanding of the mechanism of the self-assembly process in vivo. It is possible to define from in vitro studies the rate constants for the multiplicity of microscopic steps that increase the number and total mass of the different types of aggregates that are populated during this process. Such an analysis has recently been carried out for the Aβ42 peptide (4) associated with Alzheimer's disease by combining experimental and theoretical methodolo...

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“…A histidine side chain (theoretical pK a ϳ6.4), His-65, which is present in the ␤S NAC region (but not in ␣S NAC), is a likely candidate. Additionally, a previous study showed that several Asp and Glu residues, distributed throughout the N-terminal, NAC, and C-terminal domains, had altered pK a values in the ␣S monomeric state (55), suggesting that the titration of one or more of the homologous residues in ␤S could also impart the observed pH sensitivity for aggregation. The N terminus (with a theoretical pK a of 7.7 Ϯ 0.5 (56)) may have a small charge difference in this pH range, in the monomeric state.…”

Section: ␤S Forms Fibrils At Mildly Acidic Phmentioning

confidence: 98%

A pH-dependent switch promotes β-synuclein fibril formation via glutamate residues

Moriarty

Olson

Atieh

et al. 2017

Journal of Biological Chemistry

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Edited by Wolfgang Peti ␣-Synuclein (␣S) is the primary protein associated with Parkinson's disease, and it undergoes aggregation from its intrinsically disordered monomeric form to a cross-␤ fibrillar form. The closely related homolog ␤-synuclein (␤S) is essentially fibril-resistant under cytoplasmic physiological conditions. Toxic gain-of-function by ␤S has been linked to dysfunction, but the aggregation behavior of ␤S under altered pH is not wellunderstood. In this work, we compare fibril formation of ␣S and ␤S at pH 7.3 and mildly acidic pH 5.8, and we demonstrate that pH serves as an on/off switch for ␤S fibrillation. Using ␣S/␤S domain-swapped chimera constructs and single residue substitutions in ␤S, we localized the switch to acidic residues in the N-terminal and non-amyloid component domains of ␤S. Computational models of ␤S fibril structures indicate that key glutamate residues (Glu-31 and Glu-61) in these domains may be sites of pH-sensitive interactions, and variants E31A and E61A show dramatically altered pH sensitivity for fibril formation supporting the importance of these charged side chains in fibril formation of ␤S. Our results demonstrate that relatively small changes in pH, which occur frequently in the cytoplasm and in secretory pathways, may induce the formation of ␤S fibrils and suggest a complex role for ␤S in synuclein cellular homeostasis and Parkinson's disease. ␣-Synuclein (␣S),5 the primary protein component of intracytoplasmic inclusions known as Lewy bodies (LB) in Parkinson's disease (PD), is a 140-residue, predominantly monomeric, intrinsically disordered protein (IDP) (1-6). It is abundant in the cytosol but present in many organelles (7,8). The monomers of ␣S can aggregate to soluble oligomers as well as insoluble oligomers and fibrils, which are considered to be associated with the pathogenesis of PD (9). A closely related family member, ␤-synuclein (␤S) that co-localizes with ␣S, has ϳ60% sequence identity with ␣S but has not been detected in LBs of PD patients (10, 11). Instead, it has been proposed that ␤S may delay ␣S fibril formation and ameliorate ␣S toxicity in vivo by inhibiting ␣S aggregation (12-14). Furthermore, despite the high sequence similarity, ␤S itself does not form fibrils in vitro at cytoplasmic pH without facilitating agents (15). However, recent reports highlight a role for a possible toxic gain-of-function of ␤S in two different model systems (16,17), and ␤S is a component of vesicle-like lesions in the hippocampus, whose formation accompanies dementia in PD (18). In addition, two ␤S mutations, V70M and P123H, were found in sporadic and familial dementia with LBs and have been suggested to be involved in lysosomal pathology (19 -21). There is an emerging role for ␤S in the pathophysiology of PD, but the molecular determinants of this role remain poorly studied.Understanding the complex relationship of synucleins with neurodegeneration requires consideration of their structures and functions in diverse subcellular environments, beyond the cytoplasm. Altho...

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NMR determination of pK_a values in α‐synuclein

Cited by 61 publications

References 70 publications

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

A pH-dependent switch promotes β-synuclein fibril formation via glutamate residues

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NMR determination of pKa values in α‐synuclein

Cited by 61 publications

References 70 publications

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Contextual Role of a Salt Bridge in the Phage P22 Coat Protein I-Domain

Solution conditions determine the relative importance of nucleation and growth processes in α-synuclein aggregation

A pH-dependent switch promotes β-synuclein fibril formation via glutamate residues

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NMR determination of pK_a values in α‐synuclein