Peter Verdegem

Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11‐cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.

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.

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.

Rotational resonance NMR of 13C2-labelled retinal: quantitative internuclear distance determination

Rotational resonance phenomena are investigated in the solid-state magic-angle spinning NMR of all-E-[11,20-13C2]-retinal at a magnetic field of 4.7 T. We find good agreement between experiments and numerical simulations for the rotational resonance spectral peakshapes and for the rotor-driven magnetization exchange. The internuclear distance between the 13C-labelled C11 and C20 sites is determined to be 0.301 +/- 0.008 nm (from rotational resonance spectra) and 0.300 +/- 0.010 nm (from rotor-driven magnetization exchange), in agreement with the X-ray crystallographic distance of 0.296 nm. We show rotational resonance spectra which display perturbations from intermolecular homonuclear spin-spin interactions.

Synthesis of 13C-labeled carotenoids and retinoids

A three-part strategy has been developed to study molecular interactions in biological systems at the atomic level. First, isotopically labeled carotenoids and retinoids are prepared by organic total synthetic schemes with labels at predetermined atomic positions and combinations of positions. Subsequently, the labeled compounds are incorporated in the biological system. Finally, the system is studied by isotope sensitive spectroscopic techniques. In this paper, the synthesis of 10-fold 13 C-labeled retinal palmitate and b-carotene a- crustacyanin carotene for nutritional studies is discussed. Also, the scheme to label the end positions of astaxanthin and canthaxanthin with 13 C for spectroscopic investigations of a- crustacyanin with isotope labels in the chromophore is given. The synthesis of 10-methyl retinal is discussed, starting from isotopically labeled synthons obtained via schemes to 13 C- labeled natural retinal. Finally, the possibility for spectroscopic studies of caroteno and retino proteins via an expression of apoproteins by way of genetic techniques in the post-genomic era is discussed.

Changes in structure of the chromophore in the photochemical process of bovine rhodopsin as revealed by FTIR spectroscopy for hydrogen out-of-plane vibrations

The hydrogen out-of-plane bending (HOOP) vibrations were studied in the difference Fourier transform infrared spectra of lumirhodopsin and metarhodopsin I by use of a series of specifically deuterated retinal derivatives of bovine rod outer segments. The 947 cm-1 band of lumirhodopsin and the 950 cm-1 band of metarhodopsin I were assigned to the mode composed of both 11-HOOP and 12-HOOP vibrations. This result suggests that the perturbation near C12-H of the retinal in the earlier intermediate, bathorhodopsin (Palings, van den Berg, Lugtenburg and Mathies, Biochemistry, 28 (1989) 1498-1507), is extinguished in lumirhodopsin and metarhodopsin I. Unphotolyzed rhodopsin and metarhodopsin I exhibited the 14-HOOP bands in the 12-D derivatives at 901 and 886 cm-1, respectively. Lumirhodopsin, however, did not show the 14-HOOP in the 12-D derivatives. The result suggests a change in geometrical alignment of the C14-H bond in lumirhodopsin with respect to the N-H bond of the Schiff base.

CONDENSATION OF ALL-E-RETINAL

Abstract A novel base induced self condensation product of all- E -retinal is presented. The scope of the reaction is investigated with three analogous α,β-unsaturated aldehydes.

Chapter 10 The use of isotopes for probing ligand-protein interactions and ligand structure: The rhodopsin g-protein coupled membrane receptor paradigm

Publisher Summary This chapter presents some recent applications of enrichment with stable 13C isotopes and magic angle spinning (MAS) nuclear magnetic resonance (NMR) studies of rhodopsin. Comprehensive information about the electronic and spatial structure of the ligand can be obtained when bound to the membrane with isotope labeling and MAS NMR. The chapter provides an overview of the labeling and MAS NMR studies aiming at a characterization of ligand-protein interactions for rhodopsin via the determination of chemical shifts. To illustrate the capabilities of MAS NMR for studying photoproducts, shift data on the ligand–protein interactions in bathorhodopsin, structural data for the pre-discharge metarhodopsin I, and shift data for the discharged metarhodopsin II photointermediate is also reviewed. MAS NMR, in conjunction with selective isotope enrichment, is the method of choice for NMR investigations of membrane protein receptors when in the membrane in their natural environment. It is a technique for obtaining high-resolution NMR data from solids. In a MAS NMR experiment, the chemical shift anisotropy broadening of the NMR response in the solid state is suppressed by macroscopic sample rotation around an axis at the magic angle βm = 54°44′ with respect to the applied magnetic field.