A pressure-jump EPR system to monitor millisecond conformational exchange rates of spin-labeled proteins

Abstract

Site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) using nitroxide spin labels is a well-established technology for mapping site-specific secondary and tertiary structure and for monitoring conformational changes in proteins of any degree of complexity, including membrane proteins, with high sensitivity. SDSL-EPR also provides information on protein dynamics in the time scale of ps-µs using continuous wave lineshape analysis and spin lattice relaxation time methods. However, the functionally important time domain of µs-ms, corresponding to large-scale protein motions, is inaccessible to those methods. To extend SDSL-EPR to the longer time domain, the perturbation method of pressure-jump relaxation is implemented. Here, we describe a complete high-pressure EPR system at Q-band for both static pressure and millisecond-timescale pressure-jump measurements on spin-labeled proteins. The instrument enables pressure jumps both up and down from any holding pressure, ranging from atmospheric pressure to the maximum pressure capacity of the system components (~3500 bar). To demonstrate the utility of the system, we characterize a local folding-unfolding equilibrium of T4 lysozyme. The results illustrate the ability of the system to measure thermodynamic and kinetic parameters of protein conformational exchange on the millisecond timescale.

Keywords: Electron paramagnetic resonance; conformational exchange; pressure; pressure jump; protein folding; protein kinetics; protein thermodynamics.

Figure 1:

Pressure jump system design. Diagram of the pressure-jump system and its main components. Solid black connecting lines represent areas in which the pressure travels throughout the system. Dotted connecting lines indicate electrical control and data collection pathways. Green connecting lines represent compressed air lines. Red indicates the sample cell. The “Interface Module” includes the computer, the pressure-jump software interface, the DAQ I/O device, and the solid-state relays.

Figure 2:

High pressure sample cell design. The gland nut and copper ferrule provide for easy connection to HF4 fittings from High Pressure Equipment Co. Dimensions here are in inches with centimeters in parenthesis.

Figure 3:

Resonator design and housing assembly (A) Cross-section view of the three-loop–two-gap 10 mm tall resonator used in this study. (B) Disassembled and (C) assembled resonator assembly highlighting the sample guiding apparatus with silicone O-ring to hold the sample and the Rexolite end sections to enhance the magnetic field uniformity over the region of interest.

Figure 4:

Pressure jump profile and speed. Pressure traces of pressure jumps and drops of different magnitudes. The 10–90% rise time is listed for each trace. Traces are aligned such that the midpoint of each jump or drop is aligned at 0 ms.

Figure 5:

Variable-pressure CW-EPR of T4 lysozyme 118R1. (A) Crystal structure of T4 lysozyme (PDB ID: 2NTH) with the R1 side chain shown at residue 118 (magenta). Inset: structure of the R1 side chain. (B) Pressure dependence of the Q-band EPR spectra of T4L 118R1 at 300 µM; “i” and “m” denote regions of the lineshape dominated by components corresponding to immobile and mobile states, respectively. (C) The equilibrium constant determined from simulations of the variable-pressure CW EPR spectra of T4L 118R1 in 2 M urea is plotted as indicated vs. pressure. A fit to a two-state model yields the difference in free energy $\left(\text{Δ}{G}^o\right)$, change in partial molar volume $\left(\text{Δ}{\overline{V}}^o\right)$, and change in compressibility $\left(\text{Δ}{\overline{\beta }}_T\right)$ for the folded-to-denatured transition. Grey data points were not included in the fit due to inaccuracies at low pressures in this system.

Figure 6:

Examples of pressure-jump EPR relaxation profiles of T4L 118R1. Each relaxation profile is fitted with a one-phase decay function (black line) as part of a global fitting of the complete pressure jump dataset shown in Figure S3. Inset: EPR spectrum collected at 2000 bar. The black arrow indicates the field position monitored in pressure jump experiments.

Figure 7:

Pressure dependence of the relaxation time for F↔D exchange of T4L 118R1. The relaxation time constants determined from one phase decay fits to individual pressure jump relaxation profiles (black dots) are plotted vs. pressure. The red trace is a global fit to the data constrained by a two-state model of exchange.