Blot quantification

Quantification of western blots from figure 1 and figure

Commonly used FRET fluorophores promote collapse of an otherwise disordered protein

The dimensions that unfolded proteins, including intrinsically disordered proteins (IDPs), adopt in the absence of denaturant remain controversial. We developed an analysis procedure for small-angle X-ray scattering (SAXS) profiles and used it to demonstrate that even relatively hydrophobic IDPs remain nearly as expanded in water as they are in high denaturant concentrations. In contrast, as demonstrated here, most fluorescence resonance energy transfer (FRET) measurements have indicated that relatively hydrophobic IDPs contract significantly in the absence of denaturant. We use two independent approaches to further explore this controversy. First, using SAXS we show that fluorophores employed in FRET can contribute to the observed discrepancy. Specifically, we find that addition of Alexa-488 to a normally expanded IDP causes contraction by an additional 15%, a value in reasonable accord with the contraction reported in FRET-based studies. Second, using our simulations and analysis procedure to accurately extract both the radius of gyration (Rg) and end-to-end distance (Ree) from SAXS profiles, we tested the recent suggestion that FRET and SAXS results can be reconciled if the Rg and Ree are "uncoupled" (i.e., no longer simply proportional), in contrast to the case for random walk homopolymers. We find, however, that even for unfolded proteins, these two measures of unfolded state dimensions remain proportional. Together, these results suggest that improved analysis procedures and a correction for significant, fluorophore-driven interactions are sufficient to reconcile prior SAXS and FRET studies, thus providing a unified picture of the nature of unfolded polypeptide chains in the absence of denaturant

HDX–MS finds that partial unfolding with sequential domain activation controls condensation of a cellular stress marker

Bimolecular condensation plays a role in many cellular processes. Despite considerable progress, a residue-level description of condensates has been lacking as obtaining high-resolution structural information is impeded by the condensation process itself. We overcame this issue by applying hydrogen–deuterium exchange/mass spectrometry (HDX–MS) to a canonical stress granule marker protein. We propose a sequential activation model where each domain is activated at different temperatures, executes partial unfolding, and associates only with other similarly activated domains to form the condensate, a mechanism we term thermodynamic specificity. The stress marker undergoes the same structural events upon pH- or heat-induced condensation, providing a unifying molecular portrait of stress response with the marker as a central sensor across different stresses

Circular Dichroism Spectral Data

<div>Code and description relevant to files can be found on https://github.com/dad/pab1-phase-2017/blob/master/src/biophysics/CD.np</div

Dynamic Light Scattering Data

Code and description relevant to files can be found on https://github.com/dad/pab1-phase-2017/blob/master/src/biophysics/DLS.np<div></div