Abstract
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.
Highlights
Trp and Cys residues were engineered at different positions in mouse prion protein (moPrP) in such a way that they would come in contact due to relative motion of helices in the native state of the protein
In the case of W144/C199-Atto moPrP, W144 was used as the photoinduced electron transfer (PET) quencher of the Atto 655 moiety attached to the Cys residue at position 199, to report on the dynamics across helices a1 and a3 (Figure 1)
In the case of W171/C225-Atto moPrP, W144 was mutated to a Phe residue, and a Trp residue was introduced at position 171
Summary
Structural fluctuations in proteins are critical for function (Henzler-Wildman and Kern, 2007; Yang et al, 2014) and folding (Wani and Udgaonkar, 2009; Malhotra and Udgaonkar, 2016; Frauenfelder et al, 1991; Bai et al, 1995), and have a role to play in protein misfolding and aggregation (Scheibel, 2001; Elam et al, 2003; Chiti and Dobson, 2009; Singh and Udgaonkar, 2015a). It is important to determine whether aggregation-competent conformations are formed on the protein (un)folding pathway, to determine their structures, and to define the timescales of the structural fluctuations that lead to their transient accumulation. This is true in the case of the prion protein, because 85% of prion diseases occur in a sporadic manner, presumably through stochastic fluctuations in the native state of the protein.
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