TIM Protein Binding Enhancement with Increased Fluidity of Membranes

As a healthy cell undergoes apoptosis, a cell’s self-regulation of membrane fluidity and phospholipid composition of the inner and outer leaflet begin to fail. The cell membrane can no longer keep its shape and becomes fluid, or loosely packed. In addition, negatively charged phosphatidylserine [PS] is exposed on the outer leaflet; a signal to the external environment that the cell is dying. TIM [T-cell immunoglobulin and mucin domain] proteins in the external environment selectively bind to PS in the presence of calcium. The TIM protein family all possess a tryptophan, a hydrophobic and fluorescent residue, near their binding site. Using this fluorescent property, we were able to study the differences in binding affinity for TIM3 and TIM4 when the context of PS changes – i.e. increased fluidity of the cell membrane seen in dying cells. Using previous x-ray reflectivity data, the crystalline structure of TIM3 and TIM4 bound to a tightly packed membrane can be seen. The results show that TIM4’s tryptophan is satisfied in the hydrophobic core of the cell membrane when the binding event occurs, however, TIM3’s tryptophan remains in the hydrophilic exterior when bound to a tightly packed membrane. Our experiments explore the hypothesis that exposing TIM3 and TIM4 to membranes of increased fluidity will increase TIM3's binding affinity and have small impact on TIM4's.