| The intracellular lipid binding proteins (iLBP) family are found in the cells of mammals, birds, fish, amphibians and reptiles. They function to shuttle large insoluble hydrophobic molecules, including retinal, various long chain fatty acids and etc. throughout the cell around the cytosol and nucleus. The combination of their small size and relatively large binding pocket make them suitable templates in a variety of protein design applications, including the study of an innovative class of fluorescent proteins. To pursue our goals, we used human Cellular Retinol Binding Protein II (hCRBPII). We were the first group to achieve the structure of an all-trans-retinal and the first bonafide structure of retinol-bound hCRBPII.;In the course of these studies, we have discovered hCRBPII is surprisingly capable of folding as domain swapped dimer (DS), with single mutations able to shift the folding product from monomer to dimer. Structural analysis of both wild type and multiple mutant DS dimers led us to remarkable hypotheses regarding mechanism of this phenomenon, which is different from the previous studies on this family. We proposed that the N-terminal and C-terminal halves of hCRBPII are capable of at least partially folding independently, to form "open monomer." The dimer/monomer ratio depends on the relative rates of dimerization of the open monomers, versus closing of the two halves together to form the "closed monomer". In addition, by comparing structures of holo hCRBPII DS dimer variants, we identified an extremely large change in the relative orientation of the two domains upon ligand binding in dimers. This suggests the possibility that iLBP domain swapped dimers could be allosterically regulated forms of these proteins, at least in some cases. Fatty acid binding protein 5 (FABP5), another member of iLBPs, has also been reported to forms a very similar DS dimer, which makes it likely that other family members could also form DS dimers, and have physiological importance for some members of the family.;As mentioned, a new class of fluorogenic proteins was created by binding fluorophore aldehydes in the binding pocket of hCRBPII via protonated Schiff base (PSB) formation. In this new system, emission of the designed solvatochromic fluorophore is flexible based on the polarity of the environment; therefore multicolor probes can be developed. More importantly, absorption/emission wavelengths can be tuned therefor; nonspecific labeling and background fluorescence can be reduced. By now, the absorption maxima are tuned from 501nm to 705nm and emission maxima from 613 nm to 744 nm. Covering both the red and far-red fluorescence wavelength regimes. |
