In vitro, replacing KCl by potassium glutamate (KGlu), the E. coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu− in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to other stabilizers (KCl, KF, K2SO4) and destabilizers (GuHCl, GuHSCN). GuHSCN titrations at 20 °C, performed as a function of concentration of KGlu or other salt and monitored by NTL9-fluorescence, are analyzed to obtain r-values quantifying the Hofmeister salt concentration (m3)-dependence of the unfolding equilibrium constant Kobs (r-value = −dlnKobs/dm3 = (1/RT) dΔG°obs/dm3 = m-value/RT). r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model-compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 °C, we determine a KGlu r-value of 3.22 m−1, and K2SO4, KF, KCl, GuHCl and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38 and −3.00 m−1 respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values, and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 °C.

Cold unfolding of proteins is predicted by the Gibbs− Helmholtz equation and is thought to be driven by a strongly temperature-dependent interaction of protein nonpolar groups with water. Studies of the cold-unfolded state provide insight into protein energetics, partially structured states, and folding cooperativity and are of practical interest in biotechnology. However, structural characterization of the cold-unfolded state is much less extensive than studies of thermally or chemically denatured unfolded states, in large part because the midpoint of the cold unfolding transition is usually below freezing. We exploit a rationally designed point mutation (I98A) in the hydrophobic core of the C-terminal domain of the ribosomal protein L9 that allows the cold denatured state ensemble to be observed above 0 °C at near neutral pH and ambient pressure in the absence of added denaturants. A combined approach consisting of paramagnetic relaxation enhancement measurements, analysis of small-angle X-ray scattering data, all-atom simulations, and polymer theory provides a detailed description of the cold-unfolded state. Despite a globally expanded ensemble, as determined by small-angle X-ray scattering, sequence-specific medium-and long-range interactions in the cold-unfolded state give rise to deviations from homopolymer-like behavior. Our results reveal that the cold-denatured state is heterogeneous with local and long-range intramolecular interactions that may prime the folded state and also demonstrate that significant long-range interactions are compatible with expanded unfolded ensembles. The work also highlights the limitations of homopolymer-based descriptions of unfolded states of proteins.

Background:The NMDA-sensitive glutamate receptors contain disordered cytoplasmic domains that support isoformspecific signaling. Results: Proline residues dictate the conformational dynamics in disordered proteins, which were used to affect NMDA receptor activity. Conclusion:The intrinsically disordered cytoplasmic domain is involved in specific modes of NMDA receptor regulation. Significance: The underlying dynamics of protein disorder contribute to allosteric regulation.

Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions.

The temperature dependence of the overall dimensions of the denatured state ensemble (DSE) of proteins remains unclear. Some studies indicate compaction of the DSE at high temperatures, whereas others argue that dimensions do not decrease. The degree of compaction or expansion in the cold-denatured state has been less studied. To investigate the temperature dependence of unfolded state dimensions, small angle x-ray scattering measurements were performed in native buffer in the absence of denaturant for a designed point mutant of the C-terminal domain of L9, a small cooperatively folded α-β protein, at 14 different temperatures over the range of 5-60°C. The I98A mutation destabilizes the domain such that both the DSE and the folded state are populated at 25°C in the absence of denaturant or extreme pH. Thermal unfolding as well as cold unfolding can thus be observed in the absence of denaturant, allowing a direct comparison of these regimes for the same protein using the same technique. The temperature of maximal stability, T, is 30°C. There is no detectable change in R of the unfolded state as the temperature is increased above T, but a clear expansion is detected as the temperature is decreased below T. The R of the DSE populated in buffer was found to be 27.8 ± 1.7 Å at 5°C, 21.8 ± 1.9 Å at 30°C, and 21.7 ± 2.0 Å at 60°C. In contrast, no significant temperature dependence was observed for the value of R measured in 6 M guanidine hydrochloride. The small angle x-ray scattering data reported here indicate clear differences between the cold- and thermal-unfolded states and show that there is no significant compaction at elevated temperatures.