Insight into the chromophore of rhodopsin and its Meta-II photointermediate by F solid-state NMR and chemical shift tensor calculations.

19F nuclei are useful labels in solid-state NMR studies, since their chemical shift and tensor elements are very sensitive to the electrostatic and space-filling properties of their local environment. In this study we have exploited a fluorine substituent, strategically placed at the C-12-position of 11-cis retinal, the chromophore of visual rhodopsins. This label was used to explore the local environment of the chromophore in the ground state of bovine rhodopsin and its active photo-intermediate Meta II.

Coupled HOOP signature correlates with quantum yield of isorhodopsin and analog pigments.

With a quantum yield of 0.66±0.03 the photoisomerization efficiency of the visual pigment rhodopsin (11-cis⇒all-trans chromophore) is exceptionally high.

Detecting Cytosolic Peptide Delivery with the GFP Complementation Assay in the Low Micromolar Range.

Transfection of cells with a plasmid encoding for the first ten strands of the GFP protein (GFP1-10) provides the means to detect cytosolic peptide import at low micromolar concentrations. Cytosolic import of the eleventh strand of the GFP protein either by electroporation or by cell-penetrating peptide-mediated import leads to formation of the full-length GFP protein and fluorescence. An increase in sensitivity is achieved through structural modifications of the peptide and the expression of GFP1-10 as a fusion protein with mCherry.

Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification.

Structural and functional characterization of integral membrane proteins in a bilayer environment is strongly hampered by the requirement of detergents for solubilization and subsequent purification, as detergents commonly affect their structure and/or activity. Here, we describe a rapid procedure with minimal exposure to detergent to directly assemble an overexpressed integral membrane protein into soluble lipid nanodiscs prior to purification. This is exemplified with recombinant his-tagged rhodopsin, which is rapidly extracted from its host membrane and directly assembled into membrane scaffold protein (MSP) nanodiscs.

Conformational activation of visual rhodopsin in native disc membranes.

Rhodopsin is the G protein-coupled receptor (GPCR) that serves as a dim-light receptor for vision in vertebrates. We probed light-induced conformational changes in rhodopsin in its native membrane environment at room temperature using time-resolved wide-angle x-ray scattering. We observed a rapid conformational transition that is consistent with an outward tilt of the cytoplasmic portion of transmembrane helix 6 concomitant with an inward movement of the cytoplasmic portion of transmembrane helix 5.

Molecular parameters of siRNA--cell penetrating peptide nanocomplexes for efficient cellular delivery.

Cell-penetrating peptides (CPPs) are versatile tools for the intracellular delivery of various biomolecules, including siRNA. Recently, CPPs were introduced that showed greatly enhanced delivery efficiency. However, the molecular basis of this increased activity is poorly understood.

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR.

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-(13)C(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (~120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site.

The intracellular pharmacokinetics of terminally capped peptides.

With significant progress in delivery technologies, peptides and peptidomimetics are receiving increasing attention as potential therapeutics also for intracellular applications. However, analyses of the intracellular behavior of peptides are a challenge; therefore, knowledge on the intracellular pharmacokinetics of peptides is limited. So far, most research has focused on peptide degradation in the context of antigen processing, rather than on peptide stability.

Cyclopropyl and isopropyl derivatives of 11-cis and 9-cis retinals at C-9 and C-13: subtle steric differences with major effects on ligand efficacy in rhodopsin.

Retinal is the natural ligand (chromophore) of the vertebrate rod visual pigment. It occurs in either the 11-cis (rhodopsin) or the 9-cis (isorhodopsin) configuration. In its evolution to a G protein coupled photoreceptor, rhodopsin has acquired exceptional photochemical properties.

Fluoro derivatives of retinal illuminate the decisive role of the C(12)-H element in photoisomerization and rhodopsin activation.

Rhodopsin, the visual pigment of the vertebrate rod cell, is among the best investigated members of the G-protein-coupled receptor family. Within this family a unique characteristic of visual pigments is their covalently bound chromophore, 11-cis retinal, which acts as an inverse agonist. Upon illumination it can be transformed into the all-trans isomer that acts as a full agonist.

Light penetration and photoisomerization in rhodopsin studied by numerical simulations and double-quantum solid-state NMR spectroscopy.

The penetration of light into optically thick samples containing the G-protein-coupled receptor rhodopsin is studied by numerical finite-element simulations and double-quantum solid-state NMR experiments. Illumination with white light leads to the generation of the active bathorhodopsin photostate in the outer layer of the sample but generates a large amount of the side product, isorhodopsin, in the sample interior. The overall yield of bathorhodopsin is improved by using monochromatic 420 nm illumination and by mixing the sample with transparent glass beads.

Towards an interpretation of 13C chemical shifts in bathorhodopsin, a functional intermediate of a G-protein coupled receptor.

Photoisomerization of the membrane-bound light receptor protein rhodopsin leads to an energy-rich photostate called bathorhodopsin, which may be trapped at temperatures of 120 K or lower. We recently studied bathorhodopsin by low-temperature solid-state NMR, using in situ illumination of the sample in a purpose-built NMR probe. In this way we acquired (13)C chemical shifts along the retinylidene chain of the chromophore.

Double-quantum 13C nuclear magnetic resonance of bathorhodopsin, the first photointermediate in mammalian vision.

The 13C chemical shifts of the primary visual photointermediate bathorhodopsin have been observed by performing double-quantum magic-angle-spinning NMR at low temperature in the presence of illumination. Strong isomerization shifts have been observed upon the conversion of rhodopsin into bathorhodopsin.

Alpha-retinals as rhodopsin chromophores--preference for the 9-Z configuration and partial agonist activity.

The visual pigment rhodopsin, the photosensory element of the rod photoreceptor cell in the vertebrate retina, shows in combination with an endogenous ligand, 11-Z retinal, an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process.

7,8-dihydro retinals outperform the native retinals in conferring photosensitivity to visual opsin.

The visual pigment rhodopsin presents an astonishing photochemical performance. It exhibits an unprecedented quantum yield (0.67) in a highly defined and ultrafast photoisomerization process.

Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment.

In the course of our studies on the structure/function relationship of visual pigments, we have expressed the human green cone pigment in the baculovirus/insect cell expression system. Purification of the human green cone pigment, however, has so far proven to be severely hampered by the low thermal stability of this receptor in a solubilized state. In order to overcome this problem, we tested a variety of chemical compounds that have been described to improve protein stability in various applications.

Methyl substituents at the 11 or 12 position of retinal profoundly and differentially affect photochemistry and signalling activity of rhodopsin.

The C-11=C-12 double bond of the retinylidene chromophore of rhodopsin holds a central position in its light-induced photoisomerization and hence the photosensory function of this visual pigment. To probe the local environment of the HC-11=C-12H element we have prepared the 11-methyl and 12-methyl derivatives of 11-Z retinal and incorporated these into opsin to generate the rhodopsin analogs 11-methyl and 12-methyl rhodopsin. These analog pigments form with much slower kinetics and lower efficiency than the native pigment.

Accurate measurements of 13C-13C J-couplings in the rhodopsin chromophore by double-quantum solid-state NMR spectroscopy.

A new double-quantum solid-state NMR pulse sequence is presented and used to measure one-bond 13C-13C J-couplings in a set of 13C2-labeled rhodopsin isotopomers. The measured J-couplings reveal a perturbation of the electronic structure at the terminus of the conjugated chain but show no evidence for protein-induced electronic perturbation near the C11-C12 isomerization site. This work establishes NMR methodology for measuring accurate 1JCC values in noncrystalline macromolecules and shows that the measured J-couplings may reveal local electronic perturbations of mechanistic significance.

Selective interface detection: mapping binding site contacts in membrane proteins by NMR spectroscopy.

Intermolecular contact surfaces are important regions where specific interactions mediate biological function. We introduce a new magic angle spinning solid state NMR technique, dubbed "selective interface detection spectroscopy" (SIDY). In this technique, 13C-attached protons (1Hlig) are dephased by 1H-13C REDOR.

Solid-state NMR analysis of ligand--receptor interactions reveals an induced misfit in the binding site of isorhodopsin.

Rhodopsin is the photosensitive protein of the rod photoreceptor in the vertebrate retina and is a paradigm for the superfamily of G-protein-coupled receptors (GPCRs). Natural rhodopsin contains an 11-cis-retinylidene chromophore. We have prepared the 9-cis analogue isorhodopsin in a natural membrane environment using uniformly (13)C-enriched 9-cis retinal.

Constraints of the 9-methyl group binding pocket of the rhodopsin chromophore probed by 9-halogeno substitution.

Sterical constraints of the 9-methyl-binding pocket of the rhodopsin chromophore are probed using retinal analogues carrying substituents of increasing size at the 9 position (H, F, Cl, Br, CH(3), and I). The corresponding 11-Z retinals were employed to investigate formation of photosensitive pigment, and the primary photoproduct was identified by Fourier transform infrared difference spectroscopy. In addition, any effects of cumulative strain were studied by introduction of the 9-Z configuration and/or the alpha-retinal ring structure.

The ring of the rhodopsin chromophore in a hydrophobic activation switch within the binding pocket.

The current view that the beta-ionone ring of the rhodopsin chromophore vacates its binding pocket within the protein early in the photocascade has been adopted in efforts to provide structural models of photoreceptor activation. This event casts doubt on the ability of this covalently bonded ligand to participate directly in later stages involving activation of the photoreceptor and it is difficult to translate into predictions for the activation of related G protein-coupled receptors by diffusable ligands (e.g.

Expression of the candidate circadian photopigment melanopsin (Opn4) in the mouse retinal pigment epithelium.

A number of responses to light, including circadian entrainment and pupillary constriction, are preserved in mammals that lack rod and cone photoreceptors. Recent studies have demonstrated that a subset of retinal ganglion cells (RGCs) are intrinsically photosensitive, and that these RGCs project to regions of the brain associated with the regulation of the circadian clock and pupil constriction. The photopigment gene(s) that mediate these effects of irradiance remain unidentified, although melanopsin (Opn4) has emerged as a strong candidate.

Protein-induced bonding perturbation of the rhodopsin chromophore detected by double-quantum solid-state NMR.

We have obtained carbon-carbon bond length data for the functional retinylidene chromophore of rhodopsin, with a spatial resolution of 3 pm. The very high resolution was obtained by performing double-quantum solid-state NMR on a set of noncrystalline isotopically labelled bovine rhodopsin samples. We detected localized perturbations of the carbon-carbon bond lengths of the retinylidene chromophore.