Dynamics of Quaternary Structure Transitions in R-State Carbonmonoxyhemoglobin Unveiled in Time-Resolved X-ray Scattering Patterns Following a Temperature Jump

Abstract

It is well-known that tetrameric hemoglobin binds ligands cooperatively by undergoing a ligand-induced T → R quaternary structure transition, a structure-function relationship that has long served as a model system for understanding allostery in proteins. However, kinetic studies of the reverse, R → T quaternary structure transition following photolysis of carbonmonoxyhemoglobin (HbCO) reveal complex behavior that may be better explained by the presence of two different R quaternary structures coexisting in thermal equilibrium. Indeed, we report here time-resolved small- and wide-angle X-ray scattering (SAXS/WAXS) patterns of HbCO following a temperature jump that not only provide unambiguous evidence for more than one R state, but also unveil the time scale for interconversion between them. Since the time scale for the photolysis-induced R → T transition is likely different for different R-states, this structural heterogeneity must be accounted for to properly explain the kinetic heterogeneity observed in time-resolved spectroscopic studies following photolysis of HbCO.

Figure 1.

Experimental geometry for acquiring time-resolved SAXS/WAXS images. An X-ray burst (12 keV; 3% fwhm; ~500 ns; ~3 × 10¹⁰ photons) passes through a 150-μm tungsten pinhole (not shown) located ~185 mm upstream from the capillary (red), continues through a brass collimator pipe with a 150-μm aperture tip, passes through a thin-wall, 617-μm diameter capillary (Polymicro TSP530660), enters a helium-purged pyramid through a 10 mm square, 0.5 μm thick SiN window (Norcada), and impinges on the center of a 0.51 mm diameter, partially transmissive beamstop that is attached to a Kapton film ~90 mm downstream from the capillary. A peristaltic pump (not shown) circulates protein/buffer solution through the capillary, which is affxed to a slotted, BeO ceramic support that is thermoelectrically heated/cooled, and positioned by a compact, home-built, high-speed XYZ diffractometer. An IR laser pulse (Opotek Opolette 355; 1.443 μm; ~5 ns; ~1 mJ, 20.8 Hz) is directed downward through a lens tube that focuses it to an elliptical spot on the capillary (~44 mJ-mm⁻²) and produces a localized temperature jump. The scattering pattern from an X-ray pulse passing through the transiently heated solution is recorded on a large-area, high-speed X-ray detector (Rayonix MX340-HS). The scattering vector magnitude accessible with this setup [q = (4π/λ)sin θ] spans the range 0.02−5.2 Å⁻¹.

Figure 2.

Temperature-dependent SAXS/WAXS patterns are used to deduce the time-dependent temperature following a T-jump. (A) Temperature-dependent scattering patterns were acquired from aqueous buffer (150 mM NaCl, 20 mM acetate buffer, pH 4.9) over a broad range of q according to a T-ramp data acquisition protocol (see text). The intensity is reported in units of counts per pixel from a single image, with the color-coded curves representing the average of at least 6 images for each temperature. (B) Time-dependent scattering differences acquired following a 9 °C T-jump with the temperature controller set point programmed for 24 °C. ΔI corresponds to (I_t – I₋₁₀ μs), where I₋₁₀ μs represents the scattering pattern recorded with the X-ray pulse arriving 10 μs before the laser heating pulse. The amplitude of the scattering difference decreases at longer time delays as heat diffuses beyond the volume illuminated by the laser heating pulse. (C) Time-dependence of the buffer temperature within the volume probed by the X-ray beam following a 9 °C T-jump, as determined by using the curves in part A as a calibrated molecular thermometer. The temperature stability in the plateau region (t < 316 μs) was found to be 0.06 °C rms, which quantifies the precision of this “molecular thermometer.” The dashed red line corresponds to a 2-dimensional Gaussian diffusion model for thermal cooling at the laser beam center: T(t) ∝ [2π(2Dt + σ_L²)]⁻¹ where D is the diffusion constant and σ_L is laser spot size. The solid blue line corresponds to the temperature controller set point; the dashed green line corresponds to the solution temperature prior to the T-jump. Note that repetitive heating of solution in the sample capillary during T-jump measurements elevates its mean temperature about 2 °C relative to the temperature controller set point.

Figure 3.

Buffer-subtracted static SAXS/WAXS scattering of protein solution. (A) HbCO at two temperatures. (B) Hb at two temperatures. The scattering curves in panels A and B are nearly indistinguishable when plotted on a logarithmic scale. (C) Difference between high- and low-temperature curves for HbCO, from panel A, and Hb, from panel B. The observed differences are small, and arise from temperature-dependent scattering of the protein and/or its hydration shell. (D) The blue curve corresponds to the difference between the Hb (blue) and HbCO (red) curves shown in part C. This difference nominally subtracts the hydration shell contribution to the scattering difference, thereby unveiling the protein contribution to the scattering difference. The red curve corresponds to the scaled difference between the scattering curves shown in parts A and B, and it is included to illustrate the scattering difference between the quaternary conformations adopted by HbCO and Hb. Note the vertical axes in parts C and D correspond to q·ΔI, not ΔI, which facilitates visualization of both SAXS and WAXS regions on the same linear scale.

Figure 4.

Time-resolved SAXS/WAXS differences spanning 316 ns to 100 μs (8 time points per decade) following a T-jump; ΔI corresponds to (I_t – I₋₁₀ μs). (A) HbCO solution (22 °C set point; 1.3 °C offset; 13.5 °C T-jump). (B) Buffer under identical experimental conditions. (C) HbCO solution minus buffer. (D) Average scattering for the first three (blue) and last three (red) curves in the time series shown in part C. (E−H) Same as parts A−D but for Hb solution. Note the vertical axes in parts C, D, G, and H correspond to q·ΔI, not ΔI, which facilitates visualization of both SAXS and WAXS regions on the same linear scale. Over the range 0.1 < q < 1.0, the short- and long-time scattering patterns for HbCO (D) differ significantly, whereas the corresponding patterns for Hb (H) are very similar.

Figure 5.

Global analysis of time-dependent changes in the HbCO SAXS/WAXS pattern following a T-jump. (A) Time-resolved scattering curves (same as Figure 4C sans offset). (B) Scattering vectors extracted from a two-state model that reproduces the time-resolved scattering curves in part A with high fidelity. This model assumes the hydration shell contribution (blue) to the scattering difference is constant over the time range explored, and assumes the amplitude of the protein contribution (red) evolves according to first-order kinetics. For comparison, the scattering difference for Hb is shown (green). (C) Least-squares fit amplitudes of the two scattering vectors. The red solid curve corresponds to [1 - exp(−t/30 μs)] and tracks time-dependent changes of the protein scattering pattern. The blue curve is constant over time and corresponds to the hydration shell contribution to the scattering difference.