Insight into the chromophore of rhodopsin and its Meta-II photointermediate by 19F 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. In addition, the chemical shift and tensor elements of the chromophore in the free state in a membrane environment and the bound state in the protein were determined. Upon binding of the chromophore into rhodopsin and Meta II, the isotropic chemical shift changes in the opposite direction by +9.7 and -8.4 ppm, respectively. An unusually large isotropic shift difference of 35.9 ppm was observed between rhodopsin and Meta II. This partly originates in the light-triggered 11-cis to all-trans isomerization of the chromophore. The other part reflects the local conformational rearrangements in the chromophore and the binding pocket. These NMR data were correlated with the available X-ray structures of rhodopsin and Meta II using bond polarization theory. For this purpose hydrogen atoms have to be inserted and hereto a family of structures were derived that best correlated with the well-established 13C chemical shifts. Based upon these structures, a 12-F derivative was obtained that best corresponded with the experimentally determined 19F chemical shifts and tensor elements. The combined data indicate strong changes in the local environment of the C-12 position and a substantially different interaction pattern with the protein in Meta II as compared to rhodopsin.

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. This is currently explained by coherent coupling of the excited state electronic wavepacket with local vibrational nuclear modes, facilitating efficient cross-over at a conical intersection onto the photoproduct energy surface. The 9-cis counterpart of rhodopsin, dubbed isorhodopsin, has a much lower quantum yield (0.26±0.03), which, however, can be markedly enhanced by modification of the retinal chromophore (7,8-dihydro and 9-cyclopropyl derivatives). The coherent coupling in the excited state is promoted by torsional skeletal and coupled HOOP vibrational modes, in combination with a twisted conformation around the isomerization region. Since such torsion will strongly enhance the infrared intensity of coupled HOOP modes, we investigated FTIR difference spectra of rhodopsin, isorhodopsin and several analog pigments in the spectral range of isolated and coupled HCCH wags. As a result we propose that the coupled HOOP signature in these retinal pigments correlates with the distribution of torsion over counteracting segments in the retinylidene polyene chain. As such the HOOP signature can act as an indicator for the photoisomerization efficiency, and can explain the higher quantum yield of the 7,8-dihydro and 9-cyclopropyl-isorhodopsin analogs.

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. We further demonstrate that, even when the MSP was his-tagged as well, partial purification of the rhodopsin-nanodiscs could be achieved exploiting immobilized-metal chromatography. Recoveries of rhodopsin up to 80% were achieved in the purified nanodisc fraction. Over 95% of contaminating membrane protein and his-tagged MSP could be removed from the rhodopsin-nanodiscs using a single Ni2+-affinity chromatography step. This level of purification is amply sufficient for functional studies. We provide evidence that the obtained rhodopsin-nanodisc preparations are fully functional both photochemically and in their ability to bind the cognate G-protein.

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. Here, we performed a detailed analysis of the molecular and physicochemical properties of seven different siRNA-CPP nanoparticles. In addition, we determined which complexes are internalized most efficiently into the leukemia cell-line SKNO-1, and subsequently inhibited the expression of a luciferase reporter gene. We demonstrated effective complexation of siRNA for all tested CPPs, and optimal encapsulation of the siRNA was achieved at very similar molar ratios independent of peptide charge. However, CPPs with an extreme high or low overall charge proved to be exceptions, suggesting an optimal range of charge for CPP-siRNA nanoparticle formation based on opposite charge. The most active CPP (PepFect6) displayed high serum resistance but also high sensitivity to decomplexation by polyanionic macromolecules, indicating the necessity for partial decomplexation for efficient uptake. Surprisingly, CPP-siRNA complexes acquired a negative ζ-potential in the presence of serum. These novel insights shed light on the observation that cell association is necessary but not sufficient for activity and motivate new research into the nature of the nanoparticle-cell interaction. Overall, our results provide a comprehensive molecular basis for the further development of peptide-based oligonucleotide transfection agents.

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. Although the HCCH torsional twist was determined to be at least 40°, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed.

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. Here, we studied the structure-activity relationship of peptides with respect to intracellular residence time and proteolytic breakdown. The peptides comprised a collection of interaction motifs of SH2 and SH3 domains with different charge but that were of similar size and carried an N-terminal fluorescein moiety. First, we show that electroporation is a highly powerful technique to introduce peptides with different charge and hydrophobicity in uniform yields. Remarkably, the peptides differed strongly in retention of intracellular fluorescence with half-lives ranging from only 1 to more than 10 h. Residence times were greatly increased for retro-inverso peptides, demonstrating that rapid loss of fluorescence is a function of peptide degradation rather than the physicochemical characteristics of the peptide. Differences in proteolytic sensitivity were further confirmed using fluorescence correlation spectroscopy as a separation-free analytical technique to follow degradation in crude cell lysates and also in intact cells. The results provide a straightforward analytical access to a better understanding of the principles of peptide stability inside cells and will therefore greatly assist the development of bioactive peptides.

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. Illumination isomerizes the chromophore to the all-trans isomer, which acts as a full agonist. This process is extremely efficient, and there is abundant evidence that the C-9 and C-13 methyl groups of retinal play a pivotal role in this process. To examine the steric limits of the C-9 and C-13 methyl binding pocket of the binding site, we have prepared C-9 and C-13 cyclopropyl and isopropyl derivatives of its native ligands and of α-retinal at C-9. Most isopropyl analogues show very poor binding, except for 9-cis-13-isopropylretinal. Most cyclopropyl derivatives exhibit intermediate binding activity, except for 9-cis-13-cyclopropylretinal, which presents good binding activity. In general, the binding site shows preference for the 9-cis analogues over the 11-cis analogues. In fact, 13-isopropyl-9-cis-retinal acts as a superagonist after illumination. Another surprising finding was that 9-cyclopropylisorhodopsin is more like native rhodopsin with respect to spectral and photochemical properties, whereas 9-cyclopropylrhodopsin behaves more like native isorhodopsin in these aspects.

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. This photoisomerization process is extremely efficient: 2 out of 3 photons are effective, full stereoselectivity is achieved, and stereoinversion occurs within 200 fs. The mechanism behind this process is still not really understood, although the available evidence points at the twisted C(9)-C(13) segment of the 11-cis ligand as the quintessence. To further dissect the role of this segment, we have generated the 10-fluoro, 12-fluoro, and 14-fluoro analogues of rhodopsin. A fluoro substituent brings in only little more volume than hydrogen, but considerably more mass and polarizability. The analogue pigments were compared to rhodopsin with respect to their photosensitivity (quantum yield), light-induced structural transitions (UV-vis and FT-IR spectroscopy), and signaling activity (G protein activation rate). We find that 14-F substitution is quite neutral, while 10-F and 12-F substitutions exert significant but distinct effects. The 10-F pigment exhibits a quantum yield similar to that of rhodopsin (0.65) but strongly perturbed thermodynamics of the structural transitions following photoactivation and only 20% of the native signaling activity. The 12-F pigment exhibits a significantly decreased quantum yield (0.47) and signaling activity (30%) but mixed effects on the structural transitions. These properties are compared to those of the corresponding methyl derivatives. We conclude that rotation of the C(12)-H bond of the rhodopsin chromophore is a major rate-limiting factor in the photoisomerization process, while the C(10)-H moiety plays a dominant role in ligand relaxation and further rearrangements following photoactivation.

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. The implications of these findings on the interpretation of previously published rhodopsin NMR data are discussed.

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. Here we compare these results with the chemical shifts of the dark state chromophore in rhodopsin, as well as with the chemical shifts of retinylidene model compounds in solution. An earlier solid-state NMR study of bathorhodopsin found only small changes in the (13)C chemical shifts upon isomerization, suggesting only minor perturbations of the electronic structure in the isomerized retinylidene chain. This is at variance with our recent measurements which show much larger perturbations of the (13)C chemical shifts. Here we present a tentative interpretation of our NMR results involving an increased charge delocalization inside the polyene chain of the bathorhodopsin chromophore. Our results suggest that the bathochromic shift of bathorhodopsin is due to modified electrostatic interactions between the chromophore and the binding pocket, whereas both electrostatic interactions and torsional strain are involved in the energy storage mechanism of bathorhodopsin.

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. This triggers the conformational changes leading to the active state Meta(rhodopsin) II. Retinal is covalently bound to Lys-296 of the protein opsin in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 4,5-dehydro,5,6-dihydro (alpha), 5,6-dihydro or 7,8-dihydro-analogs might facilitate photoisomerization of a 9-Z and a 11-Z bond. Here we describe pigment analogs generated with bovine opsin and 11-Z or 9-Z 4,5-dehydro,5,6-dihydro-retinal that were further characterized by UV-Vis and FTIR spectroscopy. The preference of opsin for native 11-Z retinal over the 9-Z isomer is reversed in 4,5-dehydro,5,6-dihydro-retinal. 9-Z 4,5-dehydro,5,6-dihydro-retinal readily generated a photosensitive pigment. This modification has no effect on the quantum yield, but affects the Batho<-->blueshifted intermediate (BSI) equilibrium and leads to a strong decrease in the G-protein activation rate because of a downshift of the pK(a) of the Meta I<-->Meta II equilibrium.

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. This triggers the conformational changes leading to the active state Meta II of this G protein-coupled receptor. The responsible ligand, retinal, is covalently bound to Lys-296 of the protein in a protonated Schiff base. The resulting positive charge delocalization over the terminal part of the polyene chain of retinal creates a conjugation defect that upon photoexcitation moves to the opposite end of the polyene. Shortening the polyene as in 5,6-dihydro- or 7,8-dihydro analogues might facilitate photoisomerization of a 9-Z and an 11-Z bond. Here we describe pigment analogues generated with bovine opsin and 11-Z 7,8-dihydro retinal or 9-Z 7,8-dihydro retinal. Both isomers readily generate photosensitive pigments that differ remarkably in spectral properties from the native pigments. In addition, in spite of the more flexible 7,8 single bond, both analogue pigments exhibit strikingly efficient photoisomerization while largely maintaining the activity toward the G-protein. These results bear upon the activation of ligand-gated signal transducers such as G protein-coupled receptors.

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. The presence of glycerol, sucrose, trehalose and lipids during extraction improved the thermal stability of the recombinant green cone pigment up to twofold. We also analyzed the effect of mutation of residues Met208, Cys212 and Cys273 into Phe in all combinations. These mutants were designed in an attempt to increase the thermal stability by replacing weakly interacting side chains in the green pigment with their counterparts in rhodopsin with strong aromatic stacking interaction. All mutants produced wild-type levels of functional pigment, but none showed an increase in thermal stability.

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. The initial photochemistry and the signaling activity of the analog pigments were investigated by UV-vis and FTIR spectroscopy, and by a G protein activation assay. Our data indicate that the ultrafast formation of the first photointermediate is strongly perturbed by the presence of an 11-methyl substituent, but much less by a 12-methyl substituent. These results support the current concept of the mechanism of the primary photoisomerization event in rhodopsin. An important stronghold of this concept is an out-of-plane movement of the C-12H element, which is facilitated by torsion as well as extended positive charge delocalization into the C-10-C-13 segment of the chromophore. We argue that this mechanism is maintained principally with a methyl substituent at C-12. In addition, we show that both an 11-methyl and a 12-methyl substitutent perturb the photointermediate cascade and finally yield a low-activity state of the receptor. The 11-methyl pigment retains about 30% of the G protein activation rate of native rhodopsin, while the 12-methyl chromophore behaves like an inverse agonist up to at least 20 degrees C, trapping the protein in a perturbed Meta-I-like conformation. We conclude that the isomerization region of the chromophore and the spatial structure of the binding site are finely tuned, in order to achieve a high photosensory potential with an efficient pathway to a high-activity state.

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. A spin diffusion period is then used to enhance long distance 1H-1H correlations, and the results are detected by a short period of cross polarization to the 13C isotope labels. This SIDY approach allows selective observation of the interface between 13C-labeled and unlabeled moieties. We have used SIDY to probe the ligand-protein binding surface between a uniformly isotopically labeled ligand cofactor, U-13C20-11-cis-retinal, and its binding site in rhodopsin (Rho), an unlabeled, membrane-embedded G-protein coupled receptor (GPCR). The observed 1HGPCR-13Clig correlations indicate multiple close contacts between the protein and the ionone ring of the ligand, in agreement with binding studies. The polyene tail of the ligand displays fewer strong correlations in the SIDY spectrum. Some correlations can be assigned to the protein side chains lining the ligand binding site, in agreement with the crystal structure.

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. Subsequently, we have determined the complete (1)H and (13)C assignments with ultra-high field solid-state magic angle spinning NMR. The 9-cis substrate conforms to the opsin binding pocket in isorhodopsin in a manner very similar to that of the 11-cis form in rhodopsin, but the NMR data reveal an improper fit of the 9-cis chromophore in this binding site. We introduce the term "induced misfit" to describe this event. Downfield proton NMR ligation shifts (Deltasigma(lig)(H) > 1 ppm) are observed for the 16,17,19-H and nearby protons of the ionone ring and for the 9-methyl protons. They provide converging evidence for global, nonspecific steric interactions between the chromophore and protein, and contrast with the specific interactions over the entire ionone ring and its substituents detected for rhodopsin. The Deltasigma(lig)(C) pattern of the polyene chain confirms the positive charge delocalization in the polyene associated with the protonation of the Schiff base nitrogen. In line with the misalignment of the ionone ring, an additional and anomalous perturbation of the (13)C response is detected in the region of the 9-cis bond. This provides evidence for strain in the isomerization region of the polyene and supports the hypothesis that perturbation of the conjugation around the cis bond induced by the protein environment assists the selective photoisomerization.