Crystallization and X-ray analysis of the reaction center from the thermophilic green bacterium Chloroflexus aurantiacus.

The photochemical reaction center of Chloroflexus (Cf.) aurantiacus, a membrane bound pigment-protein complex, has been crystallized in the presence of monodisperse polyoxyethylene detergents. The crystals possessed a pronounced polymorphism. Three different crystal forms belonging to triclinic, monoclinic and orthorhombic space groups have been characterized by X-ray analysis. The triclinic crystal form, with unit cell dimensions of a = 88 A, b = 115 A and c = 151 A, diffracts up to 3.2 A in two directions and to 4.0 A in the third direction.

Structure and protein binding interactions of the primary donor of the Chloroflexus aurantiacus reaction center.

Soret resonance, QX resonance, and QY near-infrared Fourier transform (FT) (pre)resonance Raman spectroscopies were used to determine pigment-protein interactions of specific bacteriochlorin molecules in the reaction center from Chloroflexus aurantiacus. FT Raman spectroscopy, using 1064 nm excitation, was used to selectively obtain preresonance and resonance vibrational Raman spectra of the primary donor (P) of reaction centers (RCs) from Chloroflexus aurantiacus in the Po and P.+ states, respectively. The FT Raman spectrum of RCs in their neutral P (Po) state exhibits bands at 1605, 1632, 1648, and 1696 cm-1 which are attributable to P in its resting neutral state. Specifically, the latter three Raman bands can be assigned to the conjugated C2 acetyl and C9 keto carbonyl groups of the bacteriochlorophyll (BChl) molecules constituting P. The observation of at least three such bands is indicative of a non-monomeric nature of P, consistent with the proposal that it is a dimer of BChl molecules. The 1632 cm-1 band is consistent only with a hydrogen bonded BChl acetyl carbonyl, while the 1648 cm-1 band is assigned to a non-hydrogen bonded acetyl carbonyl. The 1696 cm-1 band is consistent only with a non-hydrogen bonded keto carbonyl group; from the unusually high intensity of this latter band compared to the others, we propose that the 1696 cm-1 band contains contributions from two keto carbonyl groups, both free of hydrogen bonds. From published protein sequence alignments of the L and M subunits of Rhodobacter (Rb.) sphaeroides and Chloroflexus aurantiacus we assign the 1632 cm-1 band as arising from the C2 acetyl carbonyl of the analogous PM constituent of P, which is hydrogen bonded to tyrosine M187 in the Chloroflexus RC, and propose a pigment-protein structural model for the primary donor of Chloroflexus aurantiacus. The FT Raman spectrum of RCs in the P degrees+ state indicates that one component of the 1696 cm-1 band has upshifted 21 cm-1 to 1717 cm-1. Compared to Rb. sphaeroides which showed a 26 cm-1 upshift for the corresponding band, the 21 cm-1 upshift indicates that the + charge is more delocalized over the P.+ species of Chloroflexus; we estimate that ca. 65% of the + charge is localized on one of the two BChl molecules of the Chloroflexus primary donor as compared to ca. 80% for Rb. sphaeroides. The consequences of the proposed structure of the Chloroflexus primary donor in terms of its Po/P.+ redox midpoint potential are discussed.

Developmental study of the expression of B50/GAP-43 in rat retina.

B50/GAP-43 has been implicated in neural plasticity, development, and regeneration. Several studies of axonally transported proteins in the optic nerve have shown that this protein is synthesized by developing and regenerating retinal ganglion cells in mammals, amphibians, and fish. However, previous studies using immunohistochemistry to localize B50/GAP-43 in retina have shown that this protein is found in the inner plexiform layer in adults. Since the inner plexiform layer contains the processes of amacrine cells, ganglion cells, and bipolar cells to determine which cells in the retina express B50/GAP-43, we have now used in situ hybridization to localize the mRNA that codes for this protein in the developing rat retina. We have found that B50/GAP-43 is expressed primarily by cells in the retinal ganglion cell layer as early as embryonic day 15, and until 3 weeks postnatal. Some cells in the inner nuclear layer, possibly a subclass of amacrine cells, also express B50/GAP-43 protein and mRNA; however, the other retinal neurons-bipolar cells, photoreceptors, and horizontal cells express little, if any, B50/GAP-43 at any stage in their development. Early in development, the protein appears in the somata and axons of ganglion cells, while later in development, B50/GAP-43 becomes concentrated in the inner plexiform layer, where it continues to be expressed in adult animals. These results are discussed in terms of previous proposals as to the functions of this molecule.