NMR and Computation Reveal a Pressure-Sensitive Folded Conformation of Trp-Cage

Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, Tc5b. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of Tc5b were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of Tc5b, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on Tc5b displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools

High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States

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

Solution Structure of the Q41N Variant of Ubiquitin as a Model for the Alternatively Folded N₂ State of Ubiquitin

It is becoming increasingly clear that proteins transiently populate high-energy excited states as a necessary requirement for function. Here, we demonstrate that rational mutation based on the characteristics of the structure and dynamics of proteins obtained from pressure experiments is a new strategy for amplifying particular fluctuations in proteins. We have previously shown that ubiquitin populates a high-energy conformer, N₂, at high pressures. Here, we show that the Q41N mutation favors N₂: high-pressure nuclear magnetic resonance (NMR) shows that N₂ is ∼70% populated in Q41N but only ∼20% populated in the wild type at ambient pressure. This allows us to characterize the structure of N₂, in which α₁-helix, the following loop, β₃-strand, and β₅-strand change their orientations relative to the remaining regions. Conformational fluctuation on the microsecond time scale, characterized by ¹⁵N spin relaxation NMR analysis, is markedly increased for these regions of the mutant. The N₂ conformers produced by high pressure and by the Q41N mutation are quite similar in both structure and dynamics. The conformational change to produce N₂ is proposed to be a novel dynamic feature beyond the known recognition dynamics of the protein. Indeed, it is orthogonal to that seen when proteins containing a ubiquitin-interacting motif bind at the hydrophobic patch of ubiquitin but matches changes seen on binding to the E2 conjugating enzyme. More generally, structural and dynamic effects of hydrodynamic pressure are shown to be useful for characterizing functionally important intermediates

Close Identity between Alternatively Folded State N₂ of Ubiquitin and the Conformation of the Protein Bound to the Ubiquitin-Activating Enzyme

We present the nuclear Overhauser effect-based structure determination of the Q41N variant of ubiquitin at 2500 bar, where the alternatively folded N₂ state is 97% populated. This allows us to characterize the structure of the “pure” N₂ state of ubiquitin. The N₂ state shows a substantial change in the orientation of strand β₅ compared to that of the normal folded N₁ state, which matches the changes seen upon binding of ubiquitin to ubiquitin-activating enzyme E1. The recognition of E1 by ubiquitin is therefore best explained by conformational selection rather than induced-fit motion