Salt-Mediated Oligomerization of the Mouse Prion Protein Monitored by Real-Time NMR

Sengupta, Ishita; Bhate, Suhas; Das, Ranabir; Udgaonkar, Jayant B.

doi:10.1016/j.jmb.2017.05.006

Cited by 27 publications

(33 citation statements)

References 105 publications

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“…Over the last two decades, the information that can be extracted from NMR observables has continuously increased owing to the development of sophisticated NMR methods combined with molecular biology protocols for sample preparation, which together have considerably enriched the NMR spectroscopist’s toolkit. The advent of these advances places NMR in the front line as one of the most valuable techniques to investigate complex protein systems under a multitude of conditions (Kay 2016 ; Sormanni et al 2017 ), which include the mapping of ligand binding sites and their affinities (Arai et al 2012 ; Teilum et al 2017 ), the presence of molecular crowders (Diniz et al 2017 ), different chemical compositions of the solution (Sengupta et al 2017 ) as well as site-directed mutations (Arbesú et al 2017 ) and local post-translational modifications (Theillet et al 2012 ) whose effects expand to the whole molecule. Such experimental design generates large and multivariable sets of NMR data.…”

Section: Introductionmentioning

confidence: 99%

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data

et al. 2018

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We present Farseer-NMR (https://git.io/vAueU), a software package to treat, evaluate and combine NMR spectroscopic data from sets of protein-derived peaklists covering a range of experimental conditions. The combined advances in NMR and molecular biology enable the study of complex biomolecular systems such as flexible proteins or large multibody complexes, which display a strong and functionally relevant response to their environmental conditions, e.g. the presence of ligands, site-directed mutations, post translational modifications, molecular crowders or the chemical composition of the solution. These advances have created a growing need to analyse those systems’ responses to multiple variables. The combined analysis of NMR peaklists from large and multivariable datasets has become a new bottleneck in the NMR analysis pipeline, whereby information-rich NMR-derived parameters have to be manually generated, which can be tedious, repetitive and prone to human error, or even unfeasible for very large datasets. There is a persistent gap in the development and distribution of software focused on peaklist treatment, analysis and representation, and specifically able to handle large multivariable datasets, which are becoming more commonplace. In this regard, Farseer-NMR aims to close this longstanding gap in the automated NMR user pipeline and, altogether, reduce the time burden of analysis of large sets of peaklists from days/weeks to seconds/minutes. We have implemented some of the most common, as well as new, routines for calculation of NMR parameters and several publication-quality plotting templates to improve NMR data representation. Farseer-NMR has been written entirely in Python and its modular code base enables facile extension.Electronic supplementary materialThe online version of this article (10.1007/s10858-018-0182-5) contains supplementary material, which is available to authorized users.

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Section: Introductionmentioning

confidence: 99%

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data

et al. 2018

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“…As a result, most HSQC peaks from well-folded S1 repeats are undetectable due to a slow global tumbling resulting from the large size, or additional µs-ms dynamics. HSQC peaks could be detected only for the very flexible regions due to the "motional narrowing" effect 37,38 .…”

Section: Solution Conformations Of Ribosomal Protein S1 and Its D3 Anmentioning

confidence: 99%

“…Remarkably, very recently it was deciphered that the oligomerization of the aggregationprone but not "completely insoluble" prion protein was also predominantly mediated by salt concentrations through both non-specific ionic strength or/and specific anion-binding to proteins 38 . Therefore, the salt-enhanced aggregation of E. coli ribosomal protein S1 is expected to follow the same mechanism previously established for "completely insoluble"…”

Section: The Roles Of Dynamics Of Inter-domain Interactions In Aggregmentioning

confidence: 99%

NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very solubleE. coliS1 ribosomal protein

Lim

Song

2017

Preprint

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Unlike mammalian aging associated with many hallmarks, E. coli aging is only significantly characterized by protein aggregation, thus offering an excellent model for addressing the relationship between protein aggregation and aging. Here we characterized conformations, unfolding and dynamics of ribosomal protein S1 and its D3/D5 domains using NMR, CD and fluorescence spectroscopy. S1 is a 557-residue modular protein containing six S1 motifs. Paradoxically, while S1 is well-folded and very soluble in vitro, it was found in various lists of aggregated E. coli proteins. Our results decipher: 1) S1 has dynamic inter-domain interactions. Strikingly, S1 and its D3/D5 domains have significantly exposed hydrophobic patches characterized by irreversible unfolding. 2) Although D5 has significantly restricted backbone motion on ps-ns time scale, it has global μs-ms conformational dynamics and particularly high “global breathing” motions. 3) D5 assumes the conserved β-barrel fold but contains large hydrophobic patches at least dynamically accessible. Taken together, our study reveals that S1 could be prone to aggregation due to significant dynamics at two levels: inter-domain interactions and individual domains, which may even render buried hydrophobic patches/cores accessible for driving aggregation. This mechanism is most likely to operate in many proteins of E. coli and other organisms including human.

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“…It is also known that residues H186 and D201 together with R155, K193 and E195 form a network of electrostatic interactions between the α2-α3 and β1-α1-β2 subdomains in monomeric PrP (Hadži et al, 2015; Hosszu et al, 2010; Singh and Udgaonkar, 2015b). The disruption of these electrostatic interactions either by a lowering of pH (Singh and Udgaonkar, 2016a) and addition of salt (Sengupta et al, 2017), or by charge reversing or neutralizing pathogenic mutations (Singh and Udgaonkar, 2016a; Singh and Udgaonkar, 2015a) facilitate misfolding and oligomer formation in vitro. It appears that separation of the α2-α3 and β1-α1-β2 subdomains must occur before conversion to the β-conformation (Eghiaian et al, 2007; Hafner-Bratkovic et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein

Sengupta

Udgaonkar

2019

eLife

Self Cite

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During pathological aggregation, proteins undergo remarkable conformational re-arrangements to anomalously assemble into a heterogeneous collection of misfolded multimers, ranging from soluble oligomers to insoluble amyloid fibrils. Inspired by fluorescence resonance energy transfer (FRET) measurements of protein folding, an experimental strategy to study site-specific misfolding kinetics during aggregation, by effectively suppressing contributions from inter-molecular FRET, is described. Specifically, the kinetics of conformational changes across different secondary and tertiary structural segments of the mouse prion protein (moPrP) were monitored independently, after the monomeric units transformed into large oligomers OL, which subsequently disaggregated reversibly into small oligomers OS at pH 4. The sequence segments spanning helices α2 and α3 underwent a compaction during the formation of OL and elongation into β-sheets during the formation of OS. The β1-α1-β2 and α2-α3 subdomains were separated, and the helix α1 was unfolded to varying extents in both OL and OS.

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Salt-Mediated Oligomerization of the Mouse Prion Protein Monitored by Real-Time NMR

Cited by 27 publications

References 105 publications

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data

Farseer-NMR: automatic treatment, analysis and plotting of large, multi-variable NMR data

NMR studies reveal that protein dynamics critically mediate aggregation of the well-folded and very solubleE. coliS1 ribosomal protein

Monitoring site-specific conformational changes in real-time reveals a misfolding mechanism of the prion protein

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