Protein aggregation appear 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.
All data generated during the study are included in the manuscript and supporting files. Source data for Figures 2,3, 5, 6 and corresponding figure supplements have been uploaded as Excel file.
- Rama Reddy Goluguri
- Sreemantee Sen
- Jayant Udgaonkar
- Jayant Udgaonkar
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
- Hannes Neuweiler, University of Würzburg, Germany
© 2019, Goluguri et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Piezo1 is a stretch-gated ion channel required for mechanosensation in many organ systems. Recent findings point to a new role for Piezo1 in the gut, suggesting that it is a sensor of microbial single-stranded RNA (ssRNA) rather than mechanical force. If true, this would redefine the scope of Piezo biology. Here, we sought to replicate the central finding that fecal ssRNA is a natural agonist of Piezo1. While we observe that fecal extracts and ssRNA can stimulate calcium influx in certain cell lines, this response is independent of Piezo1. Additionally, sterilized dietary extracts devoid of gut biome RNA show similar cell line-specific stimulatory activity to fecal extracts. Together, our data highlight potential confounds inherent to gut-derived extracts, exclude Piezo1 as a receptor for ssRNA in the gut, and support a dedicated role for Piezo channels in mechanosensing.
The auto-inhibited, super-relaxed (SRX) state of cardiac myosin is thought to be crucial for regulating contraction, relaxation, and energy conservation in the heart. We used single ATP turnover experiments to demonstrate that a dilated cardiomyopathy (DCM) mutation (E525K) in human beta-cardiac myosin increases the fraction of myosin heads in the SRX state (with slow ATP turnover), especially in physiological ionic strength conditions. We also utilized FRET between a C-terminal GFP tag on the myosin tail and Cy3ATP bound to the active site of the motor domain to estimate the fraction of heads in the closed, interacting-heads motif (IHM); we found a strong correlation between the IHM and SRX state. Negative stain electron microscopy and 2D class averaging of the construct demonstrated that the E525K mutation increased the fraction of molecules adopting the IHM. Overall, our results demonstrate that the E525K DCM mutation may reduce muscle force and power by stabilizing the auto-inhibited SRX state. Our studies also provide direct evidence for a correlation between the SRX biochemical state and the IHM structural state in cardiac muscle myosin. Furthermore, the E525 residue may be implicated in crucial electrostatic interactions that modulate this conserved, auto-inhibited conformation of myosin.