Functional and shunt states of bacteriorhodopsin resolved by 250 GHz dynamic nuclear polarization-enhanced solid-state NMR

Abstract

Observation and structural studies of reaction intermediates of proteins are challenging because of the mixtures of states usually present at low concentrations. Here, we use a 250 GHz gyrotron (cyclotron resonance maser) and cryogenic temperatures to perform high-frequency dynamic nuclear polarization (DNP) NMR experiments that enhance sensitivity in magic-angle spinning NMR spectra of cryo-trapped photocycle intermediates of bacteriorhodopsin (bR) by a factor of approximately 90. Multidimensional spectroscopy of U-(13)C,(15)N-labeled samples resolved coexisting states and allowed chemical shift assignments in the retinylidene chromophore for several intermediates not observed previously. The correlation spectra reveal unexpected heterogeneity in dark-adapted bR, distortion in the K state, and, most importantly, 4 discrete L substates. Thermal relaxation of the mixture of L's showed that 3 of these substates revert to bR(568) and that only the 1 substate with both the strongest counterion and a fully relaxed 13-cis bond is functional. These definitive observations of functional and shunt states in the bR photocycle provide a preview of the mechanistic insights that will be accessible in membrane proteins via sensitivity-enhanced DNP NMR. These observations would have not been possible absent the signal enhancement available from DNP.

Fig. 1.

The isomeric forms of the retinal chromophore in bR₅₆₈, bR₅₅₅, K–M_O. Note that the ¹⁵N of the SB is protonated in all forms except M_O.

Fig. 2.

¹⁵Nζ-¹³C15 correlation experiments provide assignments of the retinal-C15 resonance in each state: dark-adapted (A); light-adapted (B), 4 L's with residual bR₅₆₈ (C), and M_o (D). Note the downfield ¹⁵N shifts observed in the L spectrum. The temperature and wavelength of the light used for preparation of each intermediate is as follows: (A) the dark-adapted state (bR₅₅₅ and bR₅₆₈) obtained by room temperature equilibration in the dark; (B) the light-adapted state (bR₅₆₈) accumulated by 532 nm irradiation of (A) at 275 K; (C) a mixture of 4 L states, with bR₅₆₈ accumulated by 640 nm irradiation of (B) at 150 K; and (D) the early M intermediate accumulated by 532 nm irradiation of (B) at 210 K. After accumulation, all of the intermediates were cooled to 90 K for data acquisition in the dark.

Fig. 3.

¹⁵Nζ-¹³C15-¹³Cx correlation spectra that trace the connectivity of resonances in the retinylidene chromophore of bR in the dark-adapted state (A), the light-adapted state (B), the K state with residual bR₅₆₈ (C), the L states with residual bR₅₆₈ (D), and the M_o state with its deprotonated SB (E). Conditions are as specified for Fig. 2.

Fig. 4.

¹⁵Nζ-¹³Cε correlation experiments in the dark-adapted state (A), the K intermediate with residual bR₅₆₈ (B), the L intermediate with residual bR₅₆₈ (C), and the M_o state (D). Conditions are as specified for Fig. 2.

Fig. 5.

Populations and ¹³C12 chemical shifts of L substrates. (A) Volumes of the ¹⁵Nζ-C15 peaks of the mixture formed by direct red light irradiation of bR₅₆₈ at 150 K and of the mixture formed by subsequent thermal relaxation at 170 K. All L's except L₁₈₆ revert to bR₅₆₈ and thus are shunt states. Note that because cross-polarization efficiencies vary among the different intermediates, the intensity changes are nonstoichiometric. (B) Evolution of the retinal ¹³C12 chemical shift through the various photocycle intermediates. Filled symbols represent functional states, and open symbols represent shunt (i.e., L₁₆₆, L₁₇₄, and L₁₈₁) states. The squares represent data from previous work (12, 28). Note that the steric interactions at C12 that are typical of the middle and late photocycle are achieved already in the functional form of L.