Rhodopsin, a light-activated G protein coupled receptor (GPCR), has been the subject of numerous biochemical and structural investigations, serving as a model receptor for GPCRs and their activation. Herein we present the 2.3 Å resolution structure of native-source rhodopsin stabilized in a conformation competent for G protein binding. An extensive water-mediated hydrogen bond network linking the chromophore binding site to the site of G protein binding is observed, providing connections to conserved motifs essential for GPCR activation. Comparison of this extensive solvent mediated hydrogen-bonding network to the positions of ordered solvent in earlier crystallographic structures of rhodopsin photointermediates reveals both static structural and dynamic functional water-protein interactions present during the activation process. When taken with observations that solvent occupies similar positions in the structures of other GPCRs, these analyses strongly support an integral role for this dynamic ordered water network in both rhodopsin and GPCR activation.
With more than 500 crystal structures determined, serine proteases make up greater than one-third of all proteases structurally examined to date, making them among the best biochemically and structurally characterized enzymes. Despite the numerous crystallographic and biochemical studies of trypsin and related serine proteases, there are still considerable shortcomings in the understanding of their catalytic mechanism. Streptomyces erythraeus trypsin (SET) does not exhibit autolysis and crystallizes readily at physiological pH; hence, it is well suited for structural studies aimed at extending the understanding of the catalytic mechanism of serine proteases. While X-ray crystallographic structures of this enzyme have been reported, no coordinates have ever been made available in the Protein Data Bank. Based on this, and observations on the extreme stability and unique properties of this particular trypsin, it was decided to crystallize it and determine its structure. Here, the first sub-angstrom resolution structure of an unmodified, unliganded trypsin crystallized at physiological pH is reported. Detailed structural analysis reveals the geometry and structural rigidity of the catalytic triad in the unoccupied active site and comparison to related serine proteases provides a context for interpretation of biochemical studies of catalytic mechanism and activity.
Structural and biophysical studies of rhodopsin have long depended upon the ready availability of bovine retina from the meat-packing industry and the relative ease of obtaining homogenous preparations of rhodopsin in the quantities and purities necessary for such study. Herein we present a modular purification methodology employing a combination of several strategies, beginning with sucrose gradient isolation of rod outer segments (ROS) from bovine retina, detergent solubilization of ROS, selective extraction of rhodopsin starting from this detergent-solubilized ROS, and further purification via size-exclusion chromatography, resulting in a preparation of high-purity rhodopsin at high concentration suitable for crystallization or other biophysical study.
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