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 Ϸ90. 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 bR568 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.ultidimensional magic-angle spinning (MAS) solid-state NMR is a general tool in structural studies of membrane proteins that are inaccessible to crystallography and solutionstate NMR, as demonstrated by recent successful applications (1-3). But the sensitivity of these experiments is low, which becomes a significant problem when multidimensional experiments are needed to characterize systems of higher molecular weight. The sensitivity deficit is even more acute when NMR signals are further divided among multiple states, as is often the case for trapped reaction intermediates. Consequently, a 1-2 order of magnitude enhancement of NMR sensitivity is essential for applications of multidimensional MAS NMR methods to studies of reaction intermediates of membrane proteins.One approach to improving the sensitivity of NMR is dynamic nuclear polarization (DNP), in which the Ϸ660-fold greater spin polarization of unpaired electrons in a paramagnetically doped glassy matrix is transferred to nuclei before an NMR experiment (4). Here, we demonstrate that high-frequency DNP by using a stable, high-power 250 GHz microwave source (5) and an efficient, nonperturbing biradical polarizing agent (6, 7), is a potentially general approach for biological MAS NMR. A 43-fold signal enhancement from DNP, combined with operation at 90 K, yields an overall 90-fold signal enhancement over previous experiments at 183 K (8). The resulting Ϸ8,100-fold savings in acquisition time permits 2-dimensional (2D) resolution of signals from mixtures of reaction intermediates that would be impossible to observe absent the enhancement available from DNP.In bacteriorhodopsin (bR), 7 transmembrane helices surround a transport channel in whic...

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...

Light-driven proton transport in bacteriorhodopsin (BR) is initiated by photoisomerization of the retinylidene chromophore, which perturbs the hydrogen bonding network in the Schiff base region of the active site. This study aimed to identify the frequency and dipolar orientation of the N-D stretching vibrations of the Schiff base before and after photoisomerization, by means of low-temperature polarized FTIR spectroscopy of [zeta-(15)N]lysine-labeled BR in D(2)O. (15)N-shifted modes were found at 2123 and 2173 cm(-1) for BR, and at 2468 and 2495 cm(-1) for the K intermediate. The corresponding N-H stretches are at approximately 2800 cm(-1) for BR and 3350-3310 cm(-1) for the K intermediate. The shift to a 350 cm(-1) higher frequency upon photoisomerization is consistent with loss of the hydrogen bond of the Schiff base. The N-D stretch frequencies of the Schiff base in BR and the K intermediate are close to the O-D stretch frequencies of strongly hydrogen bonded water and Thr89, respectively. The angles of the dipole moments of the N-D stretches to the membrane normal were determined to be 60-65 degrees for BR and approximately 90 degrees for the K intermediate. In the case of BR, the stretch orientation is expected to deviate from the N-D bond orientation due to vibrational mixing in the hydrogen bonding network. In contrast, the data for the K intermediate suggest that the N-D group is not hydrogen bonded and orients along the membrane.

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