The hydrogen exchange behavior of native cytochrome c in low concentrations of de-naturant reveals a sequence of metastable, partially unfolded forms that occupy free energy levels reaching up to the fully unfolded state. The step from one form to another is accomplished by the unfolding of one or more cooperative units of structure. The cooperative units are entire omega loops or mutually stabilizing pairs of whole helices and loops. The partially unfolded forms detected by hydrogen exchange appear to represent the major intermediates in the reversible, dynamic unfolding reactions that occur even at native conditions and thus may define the major pathway for cytochrome c folding.Under native conditions, a small fraction of any population of protein molecules occupies each possible higher energy, partially unfolded state, including even the fully unfolded state, as described by the Boltzmann distribution. The study of these partially unfolded forms (intermediates) may illuminate the fundamental cooperative nature of protein structure and define the unfolding and refolding pathways of a protein even though the intermediates are normally invisible to measurement. The energy levels and therefore the occupation of these conformationally excited states can be manipulated by denaturants and temperature. Hydrogen exchange experiments can then determine the hydrogens exposed in each higher energy form, their rates of exchange with solvent, and their sensitivity to the perturbant. From this we can infer, respectively, the structure, the free energy, and the surface exposure of each protein form.Results for cytochrome c reveal a small sequence of distinct partially unfolded forms with progressively increasing free energy and degree of unfolding. These appear to represent the major intermediates in the unfolding and refolding pathways of cytochrome c. Hydrogen exchange theoryExchangeable amide hydrogens (NH) that are involved in hydrogen-bonded structure can exchange with solvent hydrogens only when they are transiently exposed to solvent in some kind of closed to open reaction (1-3), as indicated in Eq. 1.(1)In the almost universally observed limiting case, referred to as EX2 (for bimolecular exchange) (1), the structural opening reaction enters the rate expression as a pre-equilibrium step. The exchange rate of any hydrogen, k ex , is then determined by its chemical exchange rate in the open form, k ch , multiplied by the equilibrium opening constant, K op (Eq. 2).

Summary In eukaryotic cells, diverse stresses trigger coalescence of RNA-binding proteins into stress granules. In vitro, stress-granule-associated proteins can demix to form liquids, hydrogels, and other assemblies lacking fixed stoichiometry. Observing these phenomena has generally required conditions far removed from physiological stresses. We show that poly(A)-binding protein (Pab1 in yeast), a defining marker of stress granules, phase-separates and forms hydrogels in vitro upon exposure to physiological stress conditions. Other RNA-binding proteins depend upon low-complexity regions (LCRs) or RNA for phase separation, whereas Pab1’s LCR is not required for demixing, and RNA inhibits it. Based on unique evolutionary patterns, we create LCR mutations which systematically tune its biophysical properties and Pab1 phase separation in vitro and in vivo. Mutations which impede phase separation reduce organism fitness during prolonged stress. Poly(A)-binding protein thus acts as a physiological stress sensor, exploiting phase separation to precisely mark stress onset, a broadly generalizable mechanism.

Spectroscopic studies have identified a number of proteins that appear to retain significant residual structure under even strongly denaturing conditions. Intrinsic viscosity, hydrodynamic radii, and small-angle x-ray scattering studies, in contrast, indicate that the dimensions of most chemically denatured proteins scale with polypeptide length by means of the power-law relationship expected for random-coil behavior. Here we further explore this discrepancy by expanding the length range of characterized denatured-state radii of gyration (RG) and by reexamining proteins that reportedly do not fit the expected dimensional scaling. We find that only 2 of 28 crosslink-free, prosthetic-group-free, chemically denatured polypeptides deviate significantly from a power-law relationship with polymer length. The RG of the remaining 26 polypeptides, which range from 16 to 549 residues, are well fitted (r2 = 0.988) by a power-law relationship with a best-fit exponent, 0.598 ± 0.028, coinciding closely with the 0.588 predicted for an excluded volume random coil. Therefore, it appears that the mean dimensions of the large majority of chemically denatured proteins are effectively indistinguishable from the mean dimensions of a random-coil ensemble