Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The doublebond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.energy transduction ͉ photocycle intermediates ͉ dynamic nuclear polarization ͉ ion transport ͉ retinal protein T he light-driven ion pump, bacteriorhodopsin (bR), has been studied extensively since it was discovered in the 1970s. Its availability and stability have made it the prototypical transmembrane protein, ion pump, retinal pigment, and model for G protein-coupled receptors. As such, it has been the target of a wide variety of biophysical techniques that have garnered a great deal of information about the structure of the protein and the changes that it undergoes during its functional photocycle. Nevertheless, it remains unclear how the protein stores and channels energy to translocate ions and prevent backflow.An important feature of the pump cycle ( Fig. 1) is that the change in connectivity of the active site between the two sides of the membrane occurs midway through the photocycle (in the transition from the early M state to the late M state), long after the initial photoisomerization of the retinylidene chromophore from all-trans to 13-cis (Fig. 2), and long before the thermal reisomerization of the chromophore at the end of the photocycle. Because the change in connectivity is divorced from the major isomerization events, much attention has been directed to the process(es) that might be responsible. However, in the fuller context, the more interesting question is how the active site remains connected to the extracellular surface for so long after the photoisomerization event, and what finally releases it from that set of interactions. In this light, it is not surprising that vibrational spectroscopy finds indications of a strained chromophore in the K (1-4) and L (5-8) intermediates, and a relaxed chromophore in the N intermediate (9). Evidence of strain is also seen in magic angle spinning (MAS) NMR spectra. Furthermore, MAS NMR has pinpointed the release of this strain to the transition from early M to late M (i.e., coincident with the connectivity change) and determin...

In this communication, we report enhancements of nuclear spin polarization by dynamic nuclear polarization (DNP) in static and spinning solids at a magnetic field strength of 9T (250 GHz for g = 2 electrons, 380 MHz for 1 H). In these experiments, 1 H enhancements of up to 170 ± 50 have been observed in 1-13 C-glycine dispersed in a 60:40 glycerol/water matrix at temperatures of 20 K; in addition, we have observed significant enhancements in 15 N spectra of unoriented pf1-bacteriophage. Finally, enhancements of ~17 have been obtained in two-dimensional 13 C-13 C chemical shift correlation spectra of the amino acid U-13 C, 15 N-proline during magic angle spinning (MAS), demonstrating the stability of the DNP experiment for sustained acquisition and for quantitative experiments incorporating dipolar recoupling. In all cases, we have exploited the thermal mixing DNP mechanism with the nitroxide radical 4-amino-TEMPO as the paramagnetic dopant. These are the highest frequency DNP experiments performed to date and indicate that significant signal enhancements can be realized using the thermal mixing mechanism even at elevated magnetic fields. In large measure, this is due to the high microwave power output of the 250 GHz gyrotron oscillator used in these experiments.

In this paper, we describe a 250 GHz gyrotron oscillator, a critical component of an integrated system for magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments at 9T, corresponding to 380 MHz 1 H frequency. The 250 GHz gyrotron is the first gyro-device designed with the goal of seamless integration with an NMR spectrometer for routine DNP-enhanced NMR spectroscopy and has operated under computer control for periods of up to 21 days with a 100% duty cycle. Following a brief historical review of the field, we present studies of the membrane protein bacteriorhodopsin (bR) using DNP-enhanced multidimensional NMR. These results include assignment of active site resonances in [U-13 C, 15 N]-bR and demonstrate the utility of DNP for studies of membrane proteins. Next, we review the theory of gyro-devices from quantum mechanical and classical viewpoints and discuss the unique considerations that apply to gyrotron oscillators designed for DNP experiments. We then characterize the operation of the 250 GHz gyrotron in detail, including its long-term stability and controllability. We have measured the spectral purity of the gyrotron emission using both homodyne and heterodyne techniques. Radiation intensity patterns from the corrugated waveguide that delivers power to the NMR probe were measured using two new techniques to confirm pure mode content: a thermometric approach based on the temperaturedependent color of liquid crystalline media applied to a substrate and imaging with a pyroelectric camera. We next present a detailed study of the mode excitation characteristics of the gyrotron. Exploration of the operating characteristics of several fundamental modes reveals broadband continuous frequency tuning of up to 1.8 GHz as a function of the magnetic field alone, a feature that may be exploited in future tunable gyrotron designs. Oscillation of the 250 GHz gyrotron at the second harmonic of cyclotron resonance begins at extremely low beam currents (as low 12 mA) at frequencies between 320-365 GHz, suggesting an efficient route for the generation of even higher frequency radiation. The low starting currents were attributed to an elevated cavity Q, which is confirmed by cavity thermal load measurements. We conclude with an appendix containing a detailed description of the control system that safely automates all aspects of the gyrotron operation.