Mapping protein conformational heterogeneity under pressure with site-directed spin labeling and double electron-electron resonance

Abstract

The dominance of a single native state for most proteins under ambient conditions belies the functional importance of higher-energy conformational states (excited states), which often are too sparsely populated to allow spectroscopic investigation. Application of high hydrostatic pressure increases the population of excited states for study, but structural characterization is not trivial because of the multiplicity of states in the ensemble and rapid (microsecond to millisecond) exchange between them. Site-directed spin labeling in combination with double electron-electron resonance (DEER) provides long-range (20-80 Å) distance distributions with angstrom-level resolution and thus is ideally suited to resolve conformational heterogeneity in an excited state populated under high pressure. DEER currently is performed at cryogenic temperatures. Therefore, a method was developed for rapidly freezing spin-labeled proteins under pressure to kinetically trap the high-pressure conformational ensemble for subsequent DEER data collection at atmospheric pressure. The methodology was evaluated using seven doubly-labeled mutants of myoglobin designed to monitor selected interhelical distances. For holomyoglobin, the distance distributions are narrow and relatively insensitive to pressure. In apomyoglobin, on the other hand, the distributions reveal a striking conformational heterogeneity involving specific helices in the pressure range of 0-3 kbar, where a molten globule state is formed. The data directly reveal the amplitude of helical fluctuations, information unique to the DEER method that complements previous rate determinations. Comparison of the distance distributions for pressure- and pH-populated molten globules shows them to be remarkably similar despite a lower helical content in the latter.

Keywords: EPR; compressibility; dipolar spectroscopy.

Fig. 1.

Ribbon model of holoMb with spin-labeled sites used in DEER measurements (PDB ID code: 2MBW) (43). The spheres indicate the Cα of sites where the nitroxide spin label R1 (Inset) was introduced pairwise to measure the corresponding internitroxide distances (yellow dashed lines). Blue spheres indicate reference sites (see Results). The heme group is shown in stick representation. Arrows next to helix labels indicate the N-to-C terminus direction of each helix.

Fig. 2.

Methodology for freezing under pressure. (A) Detail of sample in the pressure bomb. A borosilicate capillary is modified by the addition of a magnetic collar near the top. A silicone piston separates the sample (orange) from the ethanol pressurization fluid (red) that fills the stainless steel pressure bomb. (B) The bomb is connected to the pressure intensifier with the lower portion immersed in dry ice/ethanol at 200 K. The sample is held at the top of the bomb using the magnet where the temperature is controlled at 298 K during pressurization. (C) Under pressure, the sample is moved quickly to the bottom of the bomb, triggering rapid cooling to 200 K. (D) The bomb is depressurized, disconnected from the pressure intensifier, and submerged in the dry ice/ethanol bath. (E) The sample capillary is transferred to liquid nitrogen (blue) for cooling to 77 K in preparation for DEER data acquisition at 80 K.

Fig. 3.

Variable-pressure DEER distance distributions for holoMb and apoMb at pH 6. (Left) Background-corrected dipolar evolutions are shown in black, with fits to the data (Methods) overlaid and color-coded by pressure. (Center and Right) The area-normalized distance distributions are shown for holoMb (Center) and apoMb (Right) for (A) 12R1/132R1, (B) 31R1/70R1, (C) 41R1/132R1, (D) 57R1/132R1, (E) 70R1/132R1, (F) 31R1/87R1, and (G) 70R1/106R1. The Inset indicates the color coding of the distance distributions and fits to the dipolar evolutions. Dipolar evolutions are vertically offset for clarity. Bold labels indicate sites in each mutant in which structural motion as a function of pressure is attributed.

Fig. 4.

DEER distance distributions for the conformational ensemble of apoMb in the native state at 0 bar and pH 6, the pH-populated MG at 0 bar and pH 4.1, and the pressure-populated MG at 2 kbar and pH 6. (Left) Background-corrected dipolar evolutions of the indicated mutants are shown in black with fits to the data (Methods) overlaid and color coded by state. (Right) The area-normalized distance distributions from are shown for (A) 12R1/132R1, (B) 31R1/70R1, (C) 41R1/132R1, (D) 57R1/132R1, (E) 70R1/132R1, (F) 31R1/87R1, and (G) 70R1/106R1. The Inset indicates the color coding of the distance distributions and fits to the dipolar evolutions. Dipolar evolutions are vertically offset for clarity. Bold labels indicate sites in each mutant in which structural motion as a function of pressure is attributed.

Fig. 5.

Models for the conformational changes of selected sequences in the transition to the pressure-populated MG of apoMb at 2 kbar, pH 6. Shown are the proposed changes relative to the native structure in particular regions. In each panel, the model of the native state is based on the crystal structure of holoMb (PDB ID code: 2MBW), except for the contiguous sequence of the F helix and N terminus of G, which is locally unfolded in the native state of apoMb and is drawn in as a disordered loop in A. This sequence is omitted from B for clarity (66). Helices are shown in cylinder representation; helices that contain reference sites are colored blue, and helices that contain sites that undergo large-amplitude motions in the pressure-populated MG are in magenta. Models for a dominant conformation in the pressure-populated MG based on DEER data at 2 kbar are superimposed and shown in red. Arrows indicate a trajectory of motion followed by each segment in the pressure-populated native-to-MG transition. (A) The sequence of helix F moves out of the heme pocket. The native-state position of the F helix is modeled to satisfy the most probable distance (20 Å) of 31R1/87R1 in apoMb at 0 bar. (B) Helix G fluctuates between the native state and a twist/tilt which inserts R1 into the heme pocket; alternatively, additional fraying of the N-terminal end of helix G at high pressure could result in the disordered sequence containing 106R1 rearranging in a number of distinct conformations that insert the nitroxide into the heme pocket. (C) Helix D fuses with the E helix in a motion that involves a concurrent rotation placing the 57R1 side chain near helix B.