Biosynthetic production of fully carbon-13 labeled retinal in E. coli for structural and functional studies of rhodopsins.

The isomerization of a covalently bound retinal is an integral part of both microbial and animal rhodopsin function. As such, detailed structure and conformational changes in the retinal binding pocket are of significant interest and are studied in various NMR, FTIR, and Raman spectroscopy experiments, which commonly require isotopic labeling of retinal. Unfortunately, the de novo organic synthesis of an isotopically-labeled retinal is complex and often cost-prohibitive, especially for large scale expression required for solid-state NMR. We present the novel protocol for biosynthetic production of an isotopically labeled retinal ligand concurrently with an apoprotein in E. coli as a cost-effective alternative to the de novo organic synthesis. Previously, the biosynthesis of a retinal precursor, ß-carotene, has been introduced into many different organisms. We extended this system to the prototrophic E. coli expression strain BL21 in conjunction with the inducible expression of a ß-dioxygenase and proteo-opsin. To demonstrate the applicability of this system, we were able to assign several new carbon resonances for proteorhodopsin-bound retinal by using fully 13C-labeled glucose as the sole carbon source. Furthermore, we demonstrated that this biosynthetically produced retinal can be extracted from E. coli cells by applying a hydrophobic solvent layer to the growth medium and reconstituted into an externally produced opsin of any desired labeling pattern.

Sparse (13)C labelling for solid-state NMR studies of P. pastoris expressed eukaryotic seven-transmembrane proteins.

We demonstrate a novel sparse (13)C labelling approach for methylotrophic yeast P. pastoris expression system, towards solid-state NMR studies of eukaryotic membrane proteins. The labelling scheme was achieved by co-utilizing natural abundance methanol and specifically (13)C labelled glycerol as carbon sources in the expression medium. This strategy improves the spectral resolution by 1.5 fold, displays site-specific labelling patterns, and has advantages for collecting long-range distance restraints for structure determination of large eukaryotic membrane proteins by solid-state NMR.

Solid-state NMR spectroscopy structure determination of a lipid-embedded heptahelical membrane protein.

Determination of structure of integral membrane proteins, especially in their native environment, is a formidable challenge in structural biology. Here we demonstrate that magic angle spinning solid-state NMR spectroscopy can be used to determine structures of membrane proteins reconstituted in synthetic lipids, an environment similar to the natural membrane. We combined a large number of experimentally determined interatomic distances and local torsional restraints to solve the structure of an oligomeric membrane protein of common seven-helical fold, Anabaena sensory rhodopsin (ASR). We determined the atomic resolution detail of the oligomerization interface of the ASR trimer, and the arrangement of helices, side chains and the retinal cofactor in the monomer.

Paramagnetic relaxation enhancement reveals oligomerization interface of a membrane protein.

Protein-protein interactions play critical roles in cellular function and oligomerization of membrane proteins is a commonly observed phenomenon. Determining the oligomerization state and defining the intermolecular interface in the bilayer is generally a difficult task. Here, we use site-specific spin labeling to demonstrate that relaxation enhancements induced by covalently attached paramagnetic tag can provide distance restraints defining the intermonomer interface in oligomers formed by a seven-helical transmembrane protein Anabaena Sensory Rhodopsin (ASR). We combine these measurements with visible CD spectroscopy and cross-linking experiments to demonstrate that ASR forms tight trimers in both detergents and lipids.

Solid-state NMR spectroscopy based atomistic view of a membrane protein unfolding pathway.

Membrane protein folding, structure, and function strongly depend on a cell membrane environment, yet detailed characterization of folding within a lipid bilayer is challenging. Studies of reversible unfolding yield valuable information on the energetics of folding and on the hierarchy of interactions contributing to protein stability. Here, we devise a methodology that combines hydrogen-deuterium (H/D) exchange and solid-state NMR (SSNMR) to follow membrane protein unfolding in lipid membranes at atomic resolution through detecting changes in the protein water-accessible surface, and concurrently monitoring the reversibility of unfolding. We obtain atomistic description of the reversible part of a thermally induced unfolding pathway of a seven-helical photoreceptor. The pathway is visualized through SSNMR-detected snapshots of H/D exchange patterns as a function of temperature, revealing the unfolding intermediate and its stabilizing factors. Our approach is transferable to other membrane proteins, and opens additional ways to characterize their unfolding and stabilizing interactions with atomic resolution.

Oligomeric Structure of Anabaena Sensory Rhodopsin in a Lipid Bilayer Environment by Combining Solid-State NMR and Long-range DEER Constraints.

Oligomerization of membrane proteins is common in nature. Here, we combine spin-labeling double electron-electron resonance (DEER) and solid-state NMR (ssNMR) spectroscopy to refine the structure of an oligomeric integral membrane protein, Anabaena sensory rhodopsin (ASR), reconstituted in a lipid environment. An essential feature of such a combined approach is that it provides structural distance restraints spanning a range of ca 3-60Å while using the same sample preparation (i.e., mutations, paramagnetic labeling, and reconstitution in lipid bilayers) for both ssNMR and DEER. Direct modeling of the multispin effects on DEER signal allowed for the determination of the oligomeric order and for obtaining long-range DEER distance restraints between the ASR trimer subunits that were used to refine the ssNMR structure of ASR. The improved structure of the ASR trimer revealed a more compact packing of helices and side chains at the intermonomer interface, compared to the structure determined using the ssNMR data alone. The extent of the refinement is significant when compared with typical helix movements observed for the active states of homologous proteins. Our combined approach of using complementary DEER and NMR measurements for the determination of oligomeric structures would be widely applicable to membrane proteins where paramagnetic tags can be introduced. Such a method could be used to study the effects of the lipid membrane composition on protein oligomerization and to observe structural changes in protein oligomers upon drug, substrate, and co-factor binding.

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR.

Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with ~17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.