Multiplex Analysis of Protein-Lipid and Protein-Membrane Protein Interactions Utilizing Silicon Photonic Microring Resonators

摘要

Reactions that occur at the cell membrane are varied and critical for many biological functions. When soluble proteins interact with the cell surface, the crowded and complex environment can act as a signaling mechanism and influence blinding and activity. Parsing out the components of the membrane that influence binding is a time consuming process. There are many different types of lipids, small molecules, and glycans that can influence binding on their own or in conjunction with one another. An ideal system to determine protein-lipid and protein-membrane protein interactions in an efficient manner requires a good membrane mimic that can be multiplexed to allow for high throughput analysis of the interactions of interest.;This dissertation focuses on the development of such a high throughput method to study soluble protein interactions with lipids and membrane proteins and the application of that method to study protein-lipid interactions that govern the blood coagulation cascade. To accomplish this, nanodiscs were integrated with silicon photonic microring resonators. Nanodiscs are easy to assemble, nanometer scale lipid bilayer discs which offer a high degree of control over lipid and membrane composition. Each nanodisc is held together by two membrane scaffold proteins that can be used as a handle for attachment to surfaces without the need to modify the lipids or proteins of interest. For high throughput capabilities, these have been interfaced with silicon photonic microring resonators. The microring resonator system can independently monitor binding interactions in real time at the surface of each of the 128 microrings on the sensor chip.;Three ways of creating nanodiscs arrays were explored. First, taking advantage of the nanodiscs' innate ability to physisorb to the silicon oxide surface of the sensor chip, nanodiscs were spotted onto the rings. With this technique, arrays of up to nine different nanodiscs were used to obtain the KD and koff values for proteins of the blood coagulation cascade. Because the protein-lipid interactions were determined using the same conditions and same nanodisc array, protein binding constants could be compared directly. The second and third methods for creating nanodisc arrays took advantage of the MSP as a handle which could be expressed with peptide tags (e.g. 6X HIS or FLAG tag) or with an exposed cysteine that could be modified with a chemical tag. From these trials, it has been shown that antibody attachment via small molecule tags on the MSP and DNA attachment via DNA modified nanodiscs offer the most promising avenues for expanding the nanodisc multiplexing toolkit.;The work presented within offers a platform for the facile and high throughput study of protein-lipid and protein-membrane protein interactions. The application of these techniques to the study of protein-lipid interactions of the blood coagulation cascade has showcased the strength of our technique and provided unique insight into the lipid dependence of membrane binding proteins of the clotting cascade.