High-Pressure Conformational Ensemble of Apomyoglobin Revealed by Double Electron-Electron Resonance

The dominance of a well-ordered native state at ambient conditions for most proteins belies the functional importance of conformational fluctuations on a wide range of time and length scales. Higher energy conformational states (excited states) may play an important functional role, yet are too sparsely populated to allow spectroscopic investigation. Perturbation techniques such as high hydrostatic pressure may be employed to increase the population of excited states for study, but structural characterization is not trivial, due to the multiplicity of states in the ensemble and rapid (s-ms) conformational exchange. The method of Site-Directed Spin Labeling (SDSL) in combination with Double Electron-Electron Resonance (DEER) is ideally suited for this purpose. DEER spectroscopy on spin-labeled protein provides long range (2-8 nm) and discrete distance distributions in heterogeneous systems with angstrom-level resolution, but must be carried out at cryogenic temperatures. In order to study the high pressure conformational ensemble of proteins, we developed a method for rapidly freezing spin-labeled proteins under pressure. This kinetically traps the high pressure equilibrium for subsequent data acquisition by DEER at atmospheric pressure and cryogenic temperature. We evaluated this methodology using seven doubly-labeled mutants of myoglobin designed to monitor discrete inter-helical distances in the protein. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure, reflecting the insensitivity of the spin-label internal motion to pressure and the absence of lowlying excited states in the energy landscape of the holo protein. On the other hand, a distinct pattern of pressure-dependent changes in apomyoglobin in the range of 0 – 3000 bar signal the appearance of a molten globule state involving increased conformational fluctuations of specific helices within an otherwise folded structure. A direct comparison of the pressure- and pH-induced molten globules reveals key differences in the amplitude of motion sampled by each helix