Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding

Sengupta, Ishita; Udgaonkar, Jayant B.

doi:10.1016/j.pep.2017.07.014

Cited by 3 publications

(7 citation statements)

References 42 publications

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“…Salt is known to trigger the aggregation of moPrP at pH 4 (Jain and Udgaonkar, 2010; Singh and Udgaonkar, 2016; Sengupta and Udgaonkar, 2017). In order to understand the effect of salt on the native state dynamics of moPrP, PET-FCS experiments were carried out in the presence of 150 mM NaCl.…”

Section: Resultsmentioning

confidence: 99%

Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein

Goluguri

Sen

Udgaonkar

2019

eLife

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Protein aggregation appears to originate from partially unfolded conformations that are sampled through stochastic fluctuations of the native protein. It has been a challenge to characterize these fluctuations, under native like conditions. Here, the conformational dynamics of the full-length (23-231) mouse prion protein were studied under native conditions, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS). The slowest fluctuations could be associated with the folding of the unfolded state to an intermediate state, by the use of microsecond mixing experiments. The two faster fluctuations observed by PET-FCS, could be attributed to fluctuations within the native state ensemble. The addition of salt, which is known to initiate the aggregation of the protein, resulted in an enhancement in the time scale of fluctuations in the core of the protein. The results indicate the importance of native state dynamics in initiating the aggregation of proteins.

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Section: Resultsmentioning

confidence: 99%

Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein

Goluguri

Sen

Udgaonkar

2019

eLife

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“…While all the unlabelled and TNB-labelled mutant variants were able to form β-rich oligomers, comparable to those formed by WT moPrP as judged by CD and DLS measurements (Sengupta and Udgaonkar, 2017), (Figure 1—figure supplement 2), the local environment around donor fluorophores W144 and W197 changed in different ways upon oligomer formation. A blue shift to 345 nm and an increase in quantum yield was observed for the W144-containing mutant variants, suggesting that the local environment of W144 was more hydrophobic in the oligomers than in the monomer (Figure 1D–F).…”

Section: Resultsmentioning

confidence: 80%

“…Trp-less moPrP was therefore used as the pseudo-WT moPrP analogue for the FRET measurements. The secondary structure and size (as estimated from CD and DLS measurements, respectively) (Sengupta and Udgaonkar, 2017) of the Trp-less moPrP oligomers were found to be comparable to that of WT moPrP (Figure 1—figure supplement 2). Moreover, Trp-less moPrP formed oligomers L and S (O L and O S ) to similar extents as did WT moPrP at pH 4 in 150 mM NaCl.…”

Section: Discussionmentioning

confidence: 87%

“…Five single Trp, single Cys-containing constructs, and a Trp-less construct were used in these experiments. The cloning, expression and purification of these proteins have been described elsewhere (Sengupta and Udgaonkar, 2017). The correct mass of all the constructs was verified by ESI-MS mass spectrometry (Appendix 1, Figure 1—figure supplement 1).…”

Section: Methodsmentioning

confidence: 99%

“…The oligomers were diluted with 1X aggregation buffer to a final concentration of 10 μM for DLS measurements (Figure 1—figure supplement 2). DLS measurements were carried out as described earlier (Sengupta and Udgaonkar, 2017).…”

Section: Methodsmentioning

confidence: 99%

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

Sengupta

Udgaonkar

2019

eLife

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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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Site-specific protein modification by genetic encoded disulfide compatible thiols

Ling

Chen

Wei

et al. 2020

Chinese Chemical Letters

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Expression and purification of single cysteine-containing mutant variants of the mouse prion protein by oxidative refolding

Cited by 3 publications

References 42 publications

Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein

Microsecond sub-domain motions and the folding and misfolding of the mouse prion protein

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

Site-specific protein modification by genetic encoded disulfide compatible thiols

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