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Probing the photointermediates of light-driven sodium ion pump KR2 by DNP-enhanced solid-state NMR
(2021)
KR2 is a light-driven sodium ion pump found in marine flavobacterium Krokinobacter Eikastus. The protein belongs to the microbial rhodopsin family, which is characterized by seven transmembrane helices and a retinal cofactor covalently bound to a conserved lysine residue through a Schiff base linkage. Specific features of KR2 and other sodium pumping rhodopsins are the NDQ motif, the N-terminal helix capping the protein at the extracellular side, and the sodium ion bound at the protomer interface in the pentameric structure. The ability to pump sodium ions was a surprising discovery since the positive charge at the Schiff base was long thought to hinder the transport of non-proton cations and the Grotthuss mechanism could not be applied to explain the Na+ transport. The photocycle of KR2 revealed by flashed photolysis and ultrafast femtosecond absorption spectroscopy consists of consecutive intermediates, named K, L, M, and O.
Here, DNP-enhanced ssNMR was used to analyze various aspects of these intermediate states. The K/L-state can be generated and trapped by in-situ illumination inside the magnet at 110 K. The trapping of L-state together with the K-state at this temperature is unexpected as this usually leads to the trapping of only K-state in bacteriorhodopsin (BR), proteorhodopsin (PR), and channelrhodopsin 2 (ChR2). This observation suggests a lower energy barrier between K- and L-state in KR2. For the O-state, the intermediate was generated by illuminating outside the magnet, followed by rapid freezing in liquid nitrogen and transfer to the magnet. Based on these procedures, the retinal conformation, and the electrostatic environment at the Schiff base in KR2 dark, K-, L- and O-intermediates were probed using 13C-labeled retinals bound to 15N-labeled KR2 by both 1D and 2D magic angle spinning (MAS) NMR experiments.
The obtained data show an all-trans retinal conformation with the distortion of 150° at H-C14-C15-H in the dark state whereas the retinal has a 13-cis, 15-anti conformation in the K- and L-state after light activation. Differences between K- and L-intermediates were observed. The retinal chemical shifts of the K-state show a large deviation from the model compound behavior between the middle and end part of the polyene chain. In the L-state, these differences are much less pronounced. These observations indicate that the light energy stored in the K-state dissipates into the protein in the subsequent photointermediate states. Furthermore, an additional shielding observed for C14 in L-state indicates the slight rotation toward a more compact 13-cis, 15-syn conformation. The distortion of the H-C14-C15-H angle in the L-state (136°) is larger than in the dark state. This twist of the retinal in the L-state would play an important role in lowering the pKa of the Schiff base, which is a prerequisite for the proton transfer from the Schiff base to the proton acceptor (D116). The electrostatic environments at the Schiff base in K- and L-states cause a de-shielding of the 15N nitrogen compared to the dark state. This indicates a stepwise stronger interaction with the counterion as the Schiff base proton moves away from the Schiff base and comes closer to the D116 in the transition from K- to L-state and approaches the proton transfer step during the M-state formation. In the O-state, the retinal was found to be in the all-trans conformation but differed to the dark state in the C13, C20, and Schiff base nitrogen chemical shifts. The largest effect (9 ppm) was observed for the Schiff base nitrogen, which could be explained by the effect of the positive charge of bound Na+ near the Schiff base in the O-state, coordinated by N112 and D116 as observed in the O-state crystal structure in the pentameric form.
The structural change at the opsin followed the retinal isomerization and the energy transfer from the chromophore to the surrounding were also investigated in this thesis using various amino acids labeling schemes. Moreover, 1H-13C hNOE in combination with CE-DNP was applied to probe the dynamics of retinylidene methyl groups and 23Na MAS NMR was employed to detect the bound sodium ion at the protomer interface in KR2 dark state.
Uncaging approach, native membrane dynamics and lipidic cubic phases in biomolecular solid-state NMR
(2019)
It was previously shown for the Escherichia coli diacylglycerol kinase (DgkA) that enzyme-reactions at the membrane interface can be monitored by solid-state NMR. However, such studies can face problems due to limited accessibility of the active sites: Natural substrates for membrane enzymes, but also ligands for membrane proteins or lipid mediators, are either partitioning into the membrane and cannot be added easily, or if soluble exhibit accessibility restrictions, as they cannot freely pass through lipid bilayers. This situation complicates quantitative kinetic analysis of biochemical processes such as enzyme activity, ligand binding, but also oligomerization or folding reactions in the membrane or at its interface under MAS NMR conditions.
To overcome these limitations the feasibility and possible advantages of the uncaging approach as a new tool for biomolecular solid-state NMR to trigger reactions by light have been explored. DgkA’s enzymatic activity, exemplary of a biochemical process on the membrane interface, was thereby triggered in situ during MAS by light-induced release of its substrates that were rendered inactive with photolabile protecting groups. To be capable of uncaging sufficient amounts of substrate during MAS to follow the enzymatic reaction via 31P real-time NMR measurements, several illumination variants including an existing illumination setup to study retinal proteins under cryogenic conditions via DNP enhanced NMR were tested. As uncaging of micromole amounts of substrates requires a higher flux compared to initiation of a photocycle in retinal proteins, a new illumination setup was built with Bruker Biospin and Leoni Fibertech. It consists of a modified MAS probe and a suitable fiber bundle, allowing to efficiently couple light from high power LEDs into a sapphire rotor containing the sample, without disturbing the magnetic field homogeneity or sample rotation. By reducing the sample volume to the illuminated area up to 60 mM ATP were released by uncaging NPE ATP to initiate DgkA’s activity in several tested membrane mimetics. These mimetics included liposomes and bicelles, which are well established in the field of biomolecular solid state NMR as well as the optically transparent lipidic cubic phase of monoolein, widely used in membrane protein crystallography, but not yet well characterized as membrane mimetic under MAS conditions. A unique and powerful but compared to time and spatial resolution often underrepresented advantage of the uncaging approach for biophysical studies has been demonstrated by successful uncaging of a non-miscible lipid substrate to trigger DgkA’s kinase reaction: Initiation of processes that cannot easily be triggered by mixing. Examples of these are reactions involving highly hydrophobic, membrane partitioning compounds including lipid substrates, ligands or interaction partners, but also oligomerization or folding of biomacromolecules. The herein performed experiments therefore serve as a first demonstration of the uncaging approach’s feasibility and compatibility with a wide variety of membrane mimetics and give a first indication of its potential for a variety of biomolecular solid state NMR experiments.
As high accessibility for solutes has been a second focus for the choice of membrane mimetics, DgkA’s activity in the lipidic cubic phases of monoacylglycerols with its two continuous networks of water channels has been further characterized. Kinetic parameters obtained from 31P real time solid state NMR experiments revealed that DgkA’s activity is similar to activities obtained in swollen cubic phases in a bath solution with wider water channels. Diffusion of ATP in a non swollen cubic phase was however strongly reduced compared to ATP in solution as diffusion measurements showed. Therefore, saturation of the enzyme required distinctly higher ATP concentrations. These results thereby underline the advantage of a non invasive and label free method like NMR to directly gain information about enzymatic reactions of immobilized enzymes in porous materials. The obtained wealth of information from 31P real time NMR experiments and biochemical assays in different membrane mimetics in presence and absence of lipid substrates and activators also provided further insight into DgkA’s enzymatic activity. It confirms ATP binding and hydrolysis in the absence of a lipid substrate, in agreement with the proposed mode of substrate binding, and allowed to estimate the in vivo relevance of previously observed ATPase activity in liposomes.
Further exploration of the cubic phase as membrane mimetic for protein solid state NMR revealed its high stability under MAS at elevated temperatures and capacity to reconstitute sufficient amounts of DgkA. Unlike monoolein, DgkA was cross-polarizable in a cubic phase and exhibited similar dynamics compared to DgkA reconstituted into liposomes, allowing to acquire the herein shown dipolar coupling based 2D protein spectra. As lipidic cubic phases are not containing phospholipids, monoacylglycerols could be especially useful as membrane mimetics for 31P correlation spectra. Initial experiments under DNP conditions, where in liposomes line broadening causes severe overlap of phospholipid signals and unspecific cross polarization highlight this aspect.
In summary, herein reported results of the experiments performed with lipidic cubic phases demonstrate that they are robust and versatile membrane mimetics. They could be of advantage for a variety of solid-state NMR experiments where either optical transparency for efficient illumination is desired, accessibility for solutes and membrane components under MAS is required, or interference of phosphorous signals of other membrane mimetics must be avoided.
In the second chapter of this thesis 1H solid-state NMR as a label free method to probe membrane order and dynamics directly within a cellular and disease relevant context was used to observe the effects of soluble epoxide hydrolase (sEH) encoding gene knock-outs on membrane dynamics. Knock-out of the sEH encoding gene changed the overall membrane dynamics in the physiological temperature range of native membranes derived from mouse brains, making the bulk membrane more dynamic. To confirm that these effects are related to the enzymatic activity of sEH, substrates and products of sEH were added to evaluate their effects on membrane dynamics. 19,20 dihydroxydocosapentaenoic acid (DHDP), a product of sEH, partially reversed the knock out phenotype in a concentration dependent manner whereas the substrate 19,20 epoxydocosapentaenoic acid did not cause any effects. As both polyunsaturated fatty acids did not show differences in phase behavior in a simple phospholipid bilayer these results provide evidence that the previously observed concentration dependent DHDP induced relocation of cholesterol away from detergent resistant lipid raft fractions is associated with alteration of membrane dynamics. Therefore, also the effect of cholesterol removal via cyclodextrin on membrane dynamics was analyzed. Removal of cholesterol led to a similar temperature profile of wild type and knock out membranes thereby supporting the hypothesis that DHDP induced relocation of cholesterol is causing altered membrane dynamics. These alterations have been shown by the lead authors of the collaborative research project to induce relocation of various membrane proteins and are involved in the development of diabetic retinopathy. Furthermore, in this context inhibition of sEH has been shown to inhibit diabetic retinopathy and proposed as target for prevention of one of the leading causes of blindness in the developed world.