In situ observation of conformational dynamics and protein ligand-substrate interactions in outer-membrane proteins with DEER/PELDOR spectroscopy

Abstract

Observation of structure and conformational dynamics of membrane proteins at high resolution in their native environments is challenging because of the lack of suitable techniques. We have developed an approach for high-precision distance measurements in the nanometer range for outer-membrane proteins (OMPs) in intact Escherichia coli and native membranes. OMPs in Gram-negative bacteria rarely have reactive cysteines. This enables in situ labeling of engineered cysteines with a methanethiosulfonate spin label (MTSL) with minimal background signals. Following overexpression of the target protein, spin labeling is performed with E. coli or isolated outer membranes (OMs) under selective conditions. The interspin distances are measured in situ, using pulsed electron-electron double resonance (PELDOR or DEER) spectroscopy. The residual background signals, which are problematic for in situ structural biology, contribute specifically to the intermolecular part of the signal and can be selectively removed to extract the desired interspin distance distribution. The initial cloning stage can take 5-7 d, and the subsequent protein expression, OM isolation, spin labeling, PELDOR experiment, and data analysis typically take 4-5 d. The described protocol provides a general strategy for observing protein ligand-substrate interactions, oligomerization, and conformational dynamics of OMPs in their native OM and intact E. coli.

Figure 1 |. Schematic view of the cell envelop of Gram-negative bacteria.

The cell envelope of Gram-negative bacteria consists of an inner membrane (IM) and an outer membrane (OM), which are separated by the periplasm. The IM is a phospholipid (PL) bilayer, whereas the OM is an asymmetric bilayer consisting of PL and lipopolysaccharide (LPS). The IM contains α-helical proteins and the OM harbors numerous β-barrel proteins (or outer membrane proteins, OMPs) including the porins, which are essential for bacterial growth or pathogenicity. The OM also contains peripherally attached lipoproteins (LP). The OMPs rarely have reactive thiols and their cysteine mutants can be labeled with MTSL in E. coli or isolated OM with minimal background labeling.

Figure 2 |. Pulse sequences for electron-electron double resonance spectroscopy (DEER/PELDOR).

(a) Echo-detected field sweep spectrum of MTSL at Q-band (34 GHz, 50 K). The positions and a schematic view of the excitation profiles for the observer (in grey) and pump pulses (in red or blue) are shown. The excitation profile for the rectangular pulse is a sinc function, whereas a shaped pulse like the sech/tanh pulse (at the bottom in d) provides a larger and uniform excitation (in dashed blue) of the spins. (b) Pulse sequence for the 4-pulse DEER. The modulation of the intensity of a refocused Hahn echo is monitored as a function of the timing of the pump pulse. (c) Pulse sequence for the 5-pulse DEER. The observer sequence is similar to the 4-pulse DEER, but are applied under a Carr-Purcell (CP) condition to prolong the observation window. The first pump pulse is fixed in time and the modulation of the observer echo intensity is monitored as a function of the timing of the second pump pulse. (d) Pulse sequence for the 7-pulse CP-PELDOR. The observer pulse sequence contains an additional π pulse accompanied with a pump pulse. Shaped sech/tanh pulses are employed to minimize the artefacts due to non-uniform excitation by the successive pump pulses. The second pump pulse is fixed in time and the echo intensity is monitored while the first and the third pump pulses are moved in equal increments in the time domain.

Figure 3 |. In-situ PELDOR in native OM.

(a) Position 188 in the second extracellular loop and the TEMPO-labeled hydroxycobalamin (TEMPO-HOCbl, 25 μM) are highlighted on the BtuB crystal structure (PDB 1NQH). The TEMPO-HOCbl was synthesized as described in Box 1. (b) Original PELDOR data obtained in native OM as indicated. The data are slightly shifted along the vertical axis for clarity. For the 188R1 mutant, the data perfectly fit into a stretched exponential decay ($d=2.2$). (c) The dipolar evolution (in yellow) obtained for 188R1/TEMPO-HOCbl PELDOR after correction for the intermolecular contribution ($d=2.5$) and the corresponding fit from Tikhonov regularization (TR) is overlaid (in black). The modulation depth (Δ) is indicated. Overall, the data suggests a two-dimensional distribution of the spins over the large cell surface and deviation of the value for $d$ (from 2.0) might be for other reasons including the membrane curvature and sample inhomogeneity. (d) The dipolar spectrum obtained with Fourier transformation (in yellow) or TR (in black) of c. Frequencies corresponding to the parallel (θ = 0) and perpendicular (θ = 90) orientations of the interspin vectors to the B₀ are indicated. (e) Interspin distance distributions obtained from TR of c.

Figure 4 |. In-situ PELDOR in E. coli.

(a) The extracellular loops carrying the positions 188 and 399 are highlighted on the BtuB crystal structure (PDB 1NQH). (b) Original PELDOR data in E. coli as indicated. For WT BtuB (which is naturally Cys-less), the data fit into a stretched exponential decay ($d=2.2$), which could not be measured longer due to the weak signal. The data are slightly shifted along the vertical axis for clarity. (c) The dipolar evolution (in yellow) obtained for the 188R1/399R1 PELDOR after correction for the intermolecular contribution ($d=2.5$) and the corresponding fit from TR (in black). The modulation depth (Δ) value is indicated. Overall, the data suggests a two-dimensional distribution of the spins over the large cell surface and deviation of the value for d (from 2.0) might be for other reasons including the membrane curvature and sample inhomogeneity. (d) The dipolar spectrum obtained with Fourier transformation (in yellow) or TR (in black) of c. (e) Interspin distance distributions obtained from TR of c. The corresponding simulation on the BtuB crystal structure (PDB 1NQH) using the MMM software is overlaid (in violet), which suggests a very good agreement between the conformations observed in the crystal structure and E. coli.

Figure 5 |. In-situ MTSL labeling of BtuB in E. coli.

(a) RT CW-EPR spectra of BtuB obtained in live E. coli after labeling with 500 μM MTSL at OD₆₀₀ = 25 for 10 min at 25 °C. (b-d) MTSL labeling of BtuB 188C-399C in E. coli at 25 °C. Spin concentrations of the E. coli (normalized to unit OD₆₀₀) are given on the y-axis. (b) Spin concentration after labeling with different MTSL concentrations. Labeling was performed at OD₆₀₀ = 25 for 10 min. (c) Spin concentration after labeling for different time intervals. MTSL labeling was performed at OD₆₀₀ = 25 with 500 μM MTSL for different times as indicated. For the zero-time point, cells were pelleted immediately after mixing with MTSL (overall, which took an additional 6–7 min including centrifugation and EPR measurement). (d) Spin concentration after labeling at different OD₆₀₀ values. Labeling was performed with 500 μM MTSL for 10 min at different OD₆₀₀ values as indicated. The inset shows a contour plot summarizing the experiments in c and d. The shaded area indicates a small window for the incubation time and the cell density under which maximal labeling can be achieved. Error bars indicate a 15% error, which is typical for spin quantification using RT CW EPR spectroscopy. Similar trends were observed between independent experiments.