Energy transformations early in the bacteriorhodopsin photocycle revealed by DNP-enhanced solid-state NMR

Abstract

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...

Mechanism of a proton pump analyzed with computer simulations

“…Moreover, the persistence of a hydrogen bond between the Schiff base and wB after proton transfer [76] is consistent with the observation from NMR that in early M the deprotonated Schiff base hydrogen bonds with a hydroxyl group [65]. In the absence of the cytoplasmic water molecule the deprotonated Schiff base can interact with the hydroxyl group of T89 [30], albeit less optimally than with the water molecule.…”

“…The stabilization of a conformer poised for proton transfer could involve the interaction between the protonated Schiff base and a water molecule. NMR indicates that in L the retinal is twisted and the Schiff base interacts strongly with the counterion; the interacting partner could be a polarized water molecule, or one of the negatively charged carboxylate groups of D85/D212 [65]. The structure of an L-like state that would explain the NMR data [65] is, however, not clear (Fig.…”

“…NMR data on the L-state intermediate at cryogenic temperatures indicated that the energy landscape is rugged; four L substates were distinguished according to their 15 N chemical shifts [65]. A single L intermediate, which with the strongest Schiff base:counterion interactions, persisted when the temperature was increased [65].…”

“…The retinal twist is used by bacteriorhodopsin to store energy upon photoisomerization [3,[61][62][63][64][65]. Retinal is directly involved in two proton-transfer reactions, the deprotonation of the Schiff base by proton transfer to the extracellular D85, and re-protonation from the cytoplasmic D96.…”

Dynamic nuclear polarization of spherical nanoparticles

“…[4][5][6][7] As pointed out recently, this ''renaissance of DNP'' combined with high magnetic fields will further grow in the coming years, enabling demanding applications with better resolution. 8,9 Materials and biological systems such as membrane or microcrystalline proteins, [10][11][12][13] fibrils, 14 ligands bound to receptors, 15 polymers, [16][17][18] surface-bound species, [19][20][21][22][23] and other low concentrated samples were studied beneficially using DNP-NMR. 13,[24][25][26] Cryogenic temperatures are required to slow down nuclear and electron relaxation and to allow an efficient polarization transfer in solid-state DNP NMR experiments.…”