Lead


Lay summary
Retinal is the photon coupling cofactor of rhodopsin in the photoreceptors of the vertebrate eye retina. The photon driven cis-trans isomerization of the 11-cis-retinal triggers a cascade of signaltransduction reactions which amplify the signal and eventually result in the visual response. Once isomerized 11-trans-retinal is no longer active and must be exchanged for fresh 11-cis-retinal. Persistent vision in vertebrates therefore depends on the continuous regeneration of 11-cis-retinal from all-trans-retinal. This regeneration is a multistep reaction called the visual cycle. The enzymes of this cycle are distributed between photoreceptor cells and adjacent retinal pigment epithelium (RPE) cells. CRALBP, the retinaldehyde-binding protein, chaperones the reaction intermediates of the RPE between the regenerating enzymes and prevents premature cis-trans isomerization. It possesses a high affinity 11-cis-retinoid binding pocket and forms protein-protein complexes with the membrane protein 11-cis-retinol dehydrogenase (RDH5), the plasma membrane adaptor protein EBP50 and possibly is involved in the formation of a retinoid-processing membrane protein complex of the RPE. Dysfunction of CRALBP has been associated with a number of severe retinopathies.We aim at the understanding the 3D structure of CRALPB and the structural alterations in clinically relevant mutants as prerequisite for a molecular explanation of the visual cycle's function, certain of its pathologies and for progress towards their therapy. Furthermore we aim at the spectroscopic characterization of the retinoid-processing membrane protein complex in RPE cells and its visualization by electron microscopy.Specific aims of this research project are: (i) X-ray structure of clinically relevant CRALBP mutants and quantitative in vitro characterization of retinoid ligand binding. (ii) Characterization of the interactions between CRALBP and regenerating enzymes. (iii) Design of a light-sensing in vitro visual cycle. (iv) Characterization of retinoid-processing membrane protein complexes. We employ X-ray crystallography, site-directed mutagenesis, spectroscopic and electron microscopic methods in order to derive structure-function relationships.CRALBP is a highly abundant 11-cis-retinoid carrier in the human retina. Probing the structure-function relationships of CRALBP with its membrane bound interaction partners may provide molecular clues to the understanding of the vertebrate visual cycle and to retinal pathologies in man. This study may aid to address the issue of visual cycle regulation in the human eye.