Channelrhodopsin-2 from Chlamydomonas reinhardtii is a lightgated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15 N-labeled channelrhodopsin-2 carrying 14,15-13 C 2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P 500 1 K-like state, the slowly decaying late intermediate P 480 4 , and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray-based structure analysis, which is required for resolving molecular mechanisms.ince their discovery (1), channelrhodopsins (ChRs) have generated enormous interest because of the rapid development of their applications in optogenetics (2-7). Commonly, ChR2 from Chlamydomonas reinhardtii (8) and its variants are used thanks to their favorable expression levels. They are the only proteins known today functioning as light-gated ion channels (Fig. 1A). Like other microbial retinal proteins, they undergo a periodic photocycle. In ChRs, this photocycle is coupled to channel opening and closing as revealed in electrophysiological recordings (8). A chimera of ChR1 and ChR2 has been crystallized to yield a structure at 2.3-Å resolution (9). However, little is known on how this coupling functions on a molecular level, and a large number of studies based on visible (10-13), IR (11,[14][15][16][17][18][19], resonance Raman spectroscopy (20, 21), and EPR spectroscopy (22, 23) has been performed to address this question.The photocycles of microbial rhodopsins are usually compared with bacteriorhodopsin, the first discovered and most studied lightdriven proton pump (24). Without any illumination, microbial retinal proteins thermally equilibrate into a dark state (25). In the case of bacteriorhodopsin, for example, this state contains a mixture of two species terme...

Nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI) are supremely important techniques with numerous applications in almost all branches of science. However, until recently, NMR methodology was limited by the time constant T1 for the decay of nuclear spin magnetization through contact with the thermal molecular environment. Long-lived states, which are correlated quantum states of multiple nuclei, have decay time constants that may exceed T1 by large factors. Here we demonstrate a nuclear long-lived state comprising two 13C nuclei with a lifetime exceeding one hour in room-temperature solution, which is around 50 times longer than T1. This behavior is well-predicted by a combination of quantum theory, molecular dynamics, and quantum chemistry. Such ultra-long-lived states are expected to be useful for the transport and application of nuclear hyperpolarization, which leads to NMR and MRI signals enhanced by up to five orders of magnitude.

The strong enhancement of NMR signals achieved by hyperpolarization decays, at best, with a time constant of a few minutes. Here, we show that a combination of long-lived singlet states, molecular design, magnetic field cycling, and specific radiofrequency pulse sequences allows repeated observation of the same batch of polarized nuclei over a period of 30 min and more. We report a recycling protocol in which the enhanced nuclear polarization achieved by dissolution-DNP is observed with full intensity and then returned to singlet order. MRI experiments may be run on a portion of the available spin polarization, while the remaining is preserved and made available for a later use. An analogy is drawn with a "spin bank" or "resealable container" in which highly polarized spin order may be deposited and retrieved.

Molecules that support (13)C singlet states with lifetimes of over 10 min in solution have been designed and synthesized. The (13)C(2) spin pairs in the asymmetric alkyne derivatives are close to magnetic equivalence, so the (13)C long-lived singlet states are stable in high magnetic field and do not require maintenance by a radiofrequency spin-locking field. We suggest a model of singlet relaxation by fluctuating chemical shift anisotropy tensors combined with leakage associated with slightly broken magnetic equivalence. Theoretical estimates of singlet relaxation rates are compared with experimental values. Relaxation due to antisymmetric shielding tensor components is significant.

Proteorhodopsins (PRs) found in marine microbes are the most abundant retinal-based photoreceptors on this planet. PR variants show high levels of environmental adaptation, as their colors are tuned to the optimal wavelength of available light. The two major green and blue subfamilies can be interconverted through a L/Q point mutation at position 105. Here we reveal the structural basis behind this intriguing color-tuning effect. High-field solid-state NMR spectroscopy was used to visualize structural changes within green PR directly within the lipid bilayer upon introduction of the green–blue L105Q mutation. The observed effects are localized within the binding pocket and close to retinal carbons C14 and C15. Subsequently, magic-angle spinning (MAS) NMR spectroscopy with sensitivity enhancement by dynamic nuclear polarization (DNP) was applied to determine precisely the retinal structure around C14–C15. Upon mutation, a significantly stretched C14–C15 bond, deshielding of C15, and a slight alteration of the retinal chain’s out-of-plane twist was observed. The L105Q blue switch therefore acts locally on the retinal itself and induces a conjugation defect between the isomerization region and the imine linkage. Consequently, the S0–S1 energy gap increases, resulting in the observed blue shift. The distortion of the chromophore structure also offers an explanation for the elongated primary reaction detected by pump–probe spectroscopy, while chemical shift perturbations within the protein can be linked to the elongation of late-photocycle intermediates studied by flash photolysis. Besides resolving a long-standing problem, this study also demonstrates that the combination of data obtained from high-field and DNP-enhanced MAS NMR spectroscopy together with time-resolved optical spectroscopy enables powerful synergies for in-depth functional studies of membrane proteins.