DNP enhanced frequency-selective TEDOR experiments in bacteriorhodopsin

Abstract

We describe a new approach to multiple (13)C-(15)N distance measurements in uniformly labeled solids, frequency-selective (FS) TEDOR. The method shares features with FS-REDOR and ZF- and BASE-TEDOR, which also provide quantitative (15)N-(13)C spectral assignments and distance measurements in U-[(13)C,(15)N] samples. To demonstrate the validity of the FS-TEDOR sequence, we measured distances in [U-(13)C,(15)N]-asparagine which are in good agreement with other methods. In addition, we integrate high frequency dynamic nuclear polarization (DNP) into the experimental protocol and use FS-TEDOR to record a resolved correlation spectrum of the Arg-(13)C(gamma)-(15)N(epsilon) region in [U-(13)C,(15)N]-bacteriorhodopsin. We resolve six of the seven cross-peaks expected based on the primary sequence of this membrane protein.

Figure 1

Pulse sequence for ¹⁵N frequency selective TEDOR transfer. Only ¹⁵N spins within the bandwidth of the selective pulses contribute to the spin dynamics under REDOR. The sequence is described in the text. The REDOR pulses are phase cycled according to the XY-8 scheme and the overall phase cycle is documented elsewhere[1]. CW microwave irradiation was employed in the version of the experiment used to acquire the DNP enhanced spectrum.

Figure 2

FS-TEDOR experiment in 10% U-[¹³C,¹⁵N]-Asparagine. We employed 100 kHz TPPM decoupling during REDOR and other periods, a 1 ms Gaussian refocusing pulse (switched between the NH₃ and NH₂ sites), 50 kHz REDOR pulses. ω_r/2π = 10 kHz

Figure 3

1D DNP enhanced ¹⁵N spectra of bR illustrating the excellent S/N available with DNP that permits observation of the ¹⁵N signal of the single Schiff Base (K216). The Arg resonances irradiated with the selective pulse are labeled and lie at ~80 ppm.

Figure 4

2D ¹⁵N-¹³C selective correlation experiment focused on the Arg-¹³C_γ-¹⁵N_ε of bR. The sequence described in Figure 1 was employed with a 1.98 ms Gaussian refocusing pulse, and TPPM decoupling optimized to reduce transverse dephasing. The REDOR mixing time of 4 ms was chosen to favor short distances (~2.4–2.5 Å). The spinning frequency was 7.576 kHz, and all DNP experiments were conducted in dark-adapted bacteriorhodopsin (90 K), as described elsewhere.