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Signaling molecules such as Activin, Sonic hedgehog, Nodal, Lefty, and Vg1 have been found to be involved in determination of left-right (L-R) asymmetry in the chick, mouse, or frog. However, a common signaling pathway has not yet been identified in vertebrates. We report that Pitx2, a bicoid-type homeobox gene expressed asymmetrically in the left lateral plate mesoderm, may be involved in determination of L-R asymmetry in both mouse and chick. Since Pitx2 appears to be downstream of lefty-1 in the mouse pathway, we examined whether mouse Lefty proteins could affect the expression of Pitx2 in the chick. Our results indicate that a common pathway from lefty-1 to Pitx2 likely exists for determination of L-R asymmetry in vertebrates.

One of the most basic and unresolved puzzles in the chemistry of vision is the mechanism regulating the absorbance of the visual photoreceptors. Rhodopsin, the rod pigment that mediates black/white vision in the human eye, absorbs at 498 nm; the three cone pigments responsible for trichromatic (color) vision absorb at 425, 530, and 560 nm, respectively. Since the chromophore in these receptors is the same protonated Schiff base of 11-cis-retinal (pSb11), the spectra of these pigments are clearly a function of the protein environment the chromophore "sees"; in other words, the spectra are tuned by the protein. [1] Three mechanisms are generally agreed to be involved in spectral tuning: 1) distortion of the chromophore as a result of steric interactions with the protein binding pocket; 2) interaction of the chromophore with the counterion balancing its positive charge; and 3) interaction of the chromophore with the remaining polar residues of the amino acids lining the binding pocket. However, the importance of these contributions could not be assessed quantitatively as long as realistic structures of the visual pigments were not available. This changed with the first X-ray crystal structure of rhodopsin, [2] now solved down to 2.2-resolution, [3] which made it possible to study the chromophore including its environment with high-quality quantum-mechanical methods. [4][5][6][7] Despite considerable insights gained from these studies, fundamental questions remain, in particular to what extent the amino acid residues of the binding pocket affect the spectrum of the chromophore.Very recently the absorption cross section of pSb11 in the gas phase was determined by analyzing fragments of photochemically excited ions.[8] Devoid of the restraining forces and charges of the protein environment, the chromophore in the gas phase or vacuum appears tailor-made for high-level quantum-mechanical calculations. It also presents a welldefined point of reference for the analysis of spectral tuning in rhodopsin. Using multiconfigurational perturbation theory we have been able to reproduce the experimental absorption maximum of the bare rhodopsin chromophore (610 nm) with high accuracy.[9] Employing the same theoretical platform we show in the following that the three contributions discussed above add up quantitatively to the experimentally observed spectral shift of the chromophore on going from the vacuum to the rhodopsin molecule. By far the largest contributor to the shift is the counterion, while the role of the polar amino acid residues of the protein pocket, contrary to general consensus, is limited to modulating the spectrum.The calculations were performed on three pSb11 model systems derived from the SCC-DFTB-refined [10] geometry of the rhodopsin binding pocket with 2.2-resolution shown in Figure 1, and they increasingly reflect the influence of the protein. Starting with the optimized vacuum structure pSb11 vac described earlier, [9] changes in the geometry corresponding to the calculated rhodopsin structure were added whic...

Density functional theory (DFT) calculations based on the self-consistent-charge tight-binding approximation have been performed to study the influence of the protein pocket on the 3-dimensional structure of the 11-cis-retinal Schiff base (SB) chromophore. Starting with an effectively planar chromophore embedded in a protein pocket consisting of the 27 next-nearest amino acids, the relaxed chromophore geometry resulting from energy optimization and molecular dynamics (MD) simulations has yielded novel insights with respect to the following questions: (i) The conformation of the beta-ionone ring. The protein pocket tolerates both conformations, 6-s-cis and 6-s-trans, with a total energy difference of 0.7 kcal/mol in favor of the former. Of the two possible 6-s-cis conformations, the one with a negative twist angle (optimized value: -35 degrees ) is strongly favored, by 3.6 kcal/mol, relative to the one in which the dihedral is positive. (ii) Out-of-plane twist of the chromophore. The environment induces a nonplanar helical deformation of the chromophore, with the distortions concentrated in the central region of the chromophore, from C10 to C13. The dihedral angle between the planes formed by the bonds from C7 to C10 and from C13 to C15 is 42 degrees. (iii) The absolute configuration of the chromophore. The dihedral angle about the C12-C13 bond is +170 degrees from planar s-cis, which imparts a positive helicity on the chromophore, in agreement with earlier considerations based on theoretical and spectroscopic evidence.

To study the origin and the effects of steric strain on the chromophore conformation in rhodopsin, we have performed quantum-mechanical calculations on the wild-type retinal chromophore and four retinal derivatives, 13-demethyl-, 10-methyl-13-demethyl-, 10-methyl-, and 9-demethylretinal. For the dynamics of the whole protein, a combined quantum mechanics/molecular mechanics method (DFTB/CHARMM) was used and for the calculation of excited-state properties the nonempirical CASSCF/CASPT2 method. After relaxation inside the protein, all chromophores show significant nonplanar distortions from C10 to C13, most strongly for 10-methylretinal and least pronounced for 9-demethylretinal. In all five cases, the dihedral angle of the C10-C11=C12-C13 bond is negative which attests to the strong chiral discrimination exerted by the protein pocket. The calculations show that the nonplanar distortion of the chromophore, including the sense of rotation, is caused by a combination of two effects: the fitting of both ends to the protein matrix which imposes a distance constraint and the bonding arrangement at the Schiff base terminus. With both the counterion Glu113 and Lys296 displaced off the plane of the chromophore, their binding to N16 exerts a torque on the chromophore. As a result, the polyene chain, from N16 to C13, is twisted in a clockwise manner against the remaining part of the chromophore, leading to a C11=C12 bond with the observed negative dihedral angle. Shifts of the absorption maxima are reproduced correctly, in particular, the red shift of the 10-methyl and the strong blue shift of the 9-demethyl analogue relative to the wild type. Calculated positive rotatory strengths of the alpha-CD bands are in agreement with the calculated absolute conformation of the mutant chromophores.