Long Distance Measurements up to 160 Å in the GroEL Tetradecamer Using Q-Band DEER EPR Spectroscopy

Abstract

Current distance measurements between spin-labels on multimeric protonated proteins using double electron-electron resonance (DEER) EPR spectroscopy are generally limited to the 15-60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin-labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin-label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q-band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface-exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.

Keywords: EPR spectroscopy; biophysics; chemical physics; spectroscopic methods; structural biology.

Figure 1

GroEL spin-labeling. Ribbon diagrams of apo GroEL (PDB 1XCK)^[8] showing a single heptameric ring (top) and two stacked heptameric rings (bottom) viewed orthogonal and parallel, respectively, to the long axis of the cavity, illustrating the positions of the spin-labels (oxygen, red spheres; other atoms, blue bonds) for R268C (left panel) and E315C (right panel). The program SCWRL4.0^[9] was used to optimize side chain positions before loading the coordinates into the MMMv2013.2^[10] program to generate rotamer probabilities for the spin-labels. In these studies, two engineered, surface-exposed cysteine mutants of GroEL were employed: R268C and E315C. (Further details of protein expression, purification, nitroxide spin-labeling and sample preparation are provided in SI) For any given spin label, there are 6 intra-ring and 7 inter-ring spin pairs (Fig. 2B). The pairwise distances between spin-labels, calculated from the crystal structure (PDB 1XCK)^[8] using the program MMMv2013.2,^[10] range from 15 to 80 Å within a heptameric ring, and from 90 to 170 Å between rings. The latter encompass a broad range of distributions centred about 100 Å and 160 Å for the R268C and E315C constructs of GroEL, respectively.

Figure 2

Impact of fractional spin-labeling on the phase memory time T_m. Q-band spin echo decay curves for fully deuterated GroEL(E315C) showing the increase in T_m accompanying a reduction in the number of spin-labeled subunits obtained by diluting MTSL through the introduction of its diamagnetic analog MTS (since the probability of a surface exposed cysteine being labelled with MTSL or MTS is essentially the same^[13]). (B)) Probability of labeling 2 (red), 2 to 3 (blue) and 2 to 4 (black) subunits of GroEL as a function of the fractional population of MTSL given my [MTSL]/([MTSL]+[MTS]). The pulse sequence employed to acquire the spin-echo decay curves in panel A was a standard two-pulse Hahn echo experiment with the echo signal recorded as a function of the echo delay time with time steps of 20 ns up to a total evolution time of 90 µs, limited by the traveling-wave tube amplifier. The length of the π/2 pulse was 12 ns, the shot repetition time was set to 20 ms, and the pulse gate time used for echo integration was 32–38 ns. All Q-band (33.8 GHz) data in this paper were acquired on a Bruker E-580 spectrometer equipped with a 150 W traveling-wave tube amplifier, a model ER5107D2 resonator, and a cryofree cooling unit operating at 50K. Sample conditions here and throughout the paper were 50 µM spin-labeled, fully deuterated GroEL 14mer, 10 mM Tris pH 8, 20 mM MgCl₂, 30%/70% (v/v) d8-glycerol/D₂O.

Figure 3

Q-band DEER measurements on fully deuterated GroEL. (A) Raw (upper panels) and background subtracted (lower panels) Q-band four-pulse DEER^[1d] echo curves recorded at 50 K on GroEL(R268C) (left panels) and GroEL(E315C) (right panels). Optimized nitroxide spin-labeling to maximize the GroEL molecules with either 2 or 3 spin labels was obtained using a 1:5 ratio of MTSL to MTS (red and blue DEER echo curves); for comparison a DEER echo curve obtained on a sample of GroEL(E315C) with 100% spin-labeling is shown in black (right panel). The DEER dipolar evolution curves shown in red (t_max = 35 and 80 µs, for the R268C and E315C samples, respectively) were obtained with flip angle reduction^[6b] to minimize dipolar truncation: the ELDOR pulse flip angles (θ) were set to 72 and 64°, respectively, corresponding to normalized modulation depths (Δ_θ/Δ_θ=180°) of 0.59 and 0.62, respectively. The DEER dipolar evolution curves shown in blue (t_max = 20 and 47 µs for the R268C and E315C samples, respectively) were obtained using an ELDOR pulse flip angle of 180°. No ELDOR pulse flip angle reduction was used for the black curve (left panel). (B) P(r) distance distributions derived from the DEER echo curves (color coding as in panel A) using the programs DD^[14] and DeerAnalysis 2013^[15] (top and bottom panels, respectively). The integrals of the P(r) distributions are shown as dashed lines, and the grey envelopes indicate the predicted P(r) distributions derived from the apo GroEL crystal structure (PDB 1XCK)^[8] using the program MMMv2013.2.^[10] Good agreement is seen between the experimental and theoretical P(r) distributions, especially when ELDOR pulse flip angle reduction is employed. In the case of DeerAnalysis, L-curves were used to select the optimal Tikhonov regularization parameter α which was set to a value of 1000. The best-fit DEER echo curves calculated from the DD analysis are shown as thin black lines in panel A. The background function used by DD is an exponential with a best-fit decay rate (see SI Fig. S4). The long 160 Å inter-ring distance for GroEL(E315C) cannot be extracted from the DEER curve recorded on fully spin-labeled and deuterated GroEL(E315C) acquired with a t_max value of 18 µs (black traces in the right-hand panels of A) as shown in SI Fig. S5. The observe and ELDOR pump pulses used for the DEER evolution curves were separated by 90 MHz with the observe π/2 and π pulses set to 12 and 24 ns, respectively. The ELDOR pulse length was set to 8 ns and the flip angle adjusted by appropriate attenuation. The pump frequency was centred at the Q-band nitroxide spectrum located at +40 MHz from the centre of the resonator frequency. The τ₁ value for the first echo-period time of 400 ns was incremented eight times in 16 ns steps to average ²H modulation; the position of the ELDOR pump pulse was incremented in steps Δt = 20, 40 and 60 ns for fully spin-labeled GroEL(E315C), sparsely-labeled GroEL(R268C) and sparsely-labeled GroEL(E315C), respectively. The bandwidth of the overcoupled resonator was ~120 MHz. The second echo period time τ₂ was set t_max + 700 ns; data collection was not extended to the full τ₂ range because of a persistent "2+1" echo perturbation of the DEER echo curves at a time of about τ₁ from the final observe π pulse. The pulse gate time used for echo integration was 32–38 ns. Total acquisition time was ~24 hours for the shorter t_max values, and ~4 days for the t_max = 80 µs data.