Thermodynamic Stability and Folding Kinetics of Membrane Proteins

Constituting a significant fraction of human proteome, membrane proteins participate in almost every aspect of the biological functions. As a result, mutations that cause misfolding or destabilization of membrane proteins are associated with many human diseases. Because of these crucial roles in vivo, many membrane proteins become important pharmaceutical targets. To develop potential therapeutics for these diseases, it is therefore essential to understanding the fundamental folding and stability properties of membrane proteins. Despite of the importance, the progress of research on membrane protein folding and stability is still far behind of that on soluble proteins. Main challenges arise from the difficulties of obtaining membrane proteins in homogeneity, heterogeneous lipid bilayer environments, and lack of tools that can systematically study their folding and stability. We have developed a novel technique called steric trapping that designed to investigate the thermodynamic stability of membrane protein in lipid bilayer-like conditions and were applied to several membrane proteins. With the success of developing the method, we are moving forward to develop the second generation of the method which can address some disadvantages that steric trapping has faced. In this proposal, we construct this new “clipped trapping system” which can then be a complementary tool to steric trapping to be employed on more membrane protein studies. In Aim 1, I outline the steps of the construction of clipped trapping system based on a similar concept of steric trapping. Detailed structural information and Cys residues are required for using steric trapping which limit its generalization to be used on other membrane proteins. The newly developed clipped trapping method can resolve these issues. I will use this method on bacteriorhodopsin again and a membrane protein with unknown detailed structure to characterize their thermodynamic stability and to prove its feasibility. In the second aim, folding and unfolding kinetic experiments are designed using clipped trapping method. We plan to measure the folding and unfolding rate constants of bR under bilayer-like environment. In the final aim, I want to more quantitatively study how the protein degradation rate in vivo correlates to the changes of thermodynamic stability of membrane protein measured in vitro. I will use the in vitro clipped trapping system reconstituted into proteoliposome conditions, combining with a well-developed in vivo global protein stability profiling assay to address any possible correlation. In summary, the newly developed clipped trapping method will give us extra tool to study biophysical properties of membrane proteins in desired conditions. The results from the proposal will provide not only the general properties of folding stability and kinetics of the membrane protein that one is interested in, but also the insight of how stability changes of membrane proteins affect their biological processes in cell.