I. Crystallization of CRBPII mutants as a probe for understanding wavelength regulation. II. Reengineering and crystallization of Cellular Retinol Binding Protein II (CRBPII) as a fluorescent tag. III. Synthesis of gamma-carboxy glutamic acid and applications towards the synthesis of conantokins.

摘要

I. The mechanism by which humans are able to see colors has been an intriguing area of study over the past several decades. This interest comes from the fact that the different opsins bind to a single chromophore, retinal, as a protonated Schiff base and a wavelength range of 420 nm to 560 nm is observed. Different postulations have been put forward and mutagenic studies have been done on rhodopsin in an attempt to explain this phenomenon. Without crystallographic evidence, the results of these experiments proved to be inconclusive. Rhodopsin being a trans-membrane protein is very difficult to crystallize and give poor expression yields. As a result our lab has engineered a small cytosolic protein, Cellular Retinol Binding Protein II (CRBPII), as a rhodopsin mimic. Our studies, with the aid of high resolution crystal structures, have shown that chromophore conformation and complete burial of the chromophore in the binding pocket is essential for wavelength regulation.;II. Fluorescent proteins tags are widely used in the field of biological sciences. Fluorescence based assays provide a means of probing protein localization, protein-protein interactions, protein expression and other biological processes without isolation from the cellular environment. Well renown Green Fluorescent Protein (GFP) and modified GFP's have been developed and are currently widely used as fluorescent tags. There are also non-protein fluorescent tags that are also currently being widely used; these include the SNAP and FLAsH tags. Even with the advent of these fluorophoric protein tags there is still a lack of red and near-IR fluorescent proteins that are bright and photochemically stable. Since Cellular Retinol Binding Protein II (CRBPII), has proven to be a robust system and binds all-trans-retinal in the nanomolar range, we decided to consider other molecules (fluorophores) that would bind as a protonated Schiff base in order to develop a new red fluorescent protein tag. Several chromophores have been used but to date two of them, Merocyanine and Julolidine, has proven to be suitable. We were able to get high resolution crystal structures of CRBPII with Merocyanine, which provided a platform for analyzing the protein-fluorophore interactions to improve the quantum efficiency of the system.;III. Gamma-carboxyglutamic acid (Gla) is formed in humans as a post-translational modification of glutamic acid via the vitamin D carboxylase enzyme. This modification allows for tight binding of calcium ions, which allows for calciferation of bones. This modified amino acid is also essential in the blood coagulation cascade processes. The Gla residues are also found in a family of peptides known as the conantokins, which are found in the venom of sea snails of the Conus genus. The metallo-zipper motif was first discovered in these conantokins. The 'metallo-zipper' motif provides a novel interface, which is dependent only on metal chelation. It was found that in the presence of metal ions such as Ca2+, Mg2+, Zn2+, and Cu2+, the structure of both Con-G and Con-T is changed from a linear structure to a helical structure. We have devised a new synthesis of gla and made attempts to synthesize different variants of the conantokins.