Computational and Experimental Investigations of Folding Principles and Design of Outer Membrane Proteins

Outer membrane proteins are found in the outer membrane of Gram-negative bacteria, mitochondria and chloroplasts, and serve essential functions for cell viability. They are also important therapeutic targets of developing drugs, and are the focuses of development of single-molecule sensors. Despite their importance, our understanding of outer membrane protein folding as well as outer membrane protein design have lagged far behind compared to soluble proteins due to the difficulties in studying them in their natural form. Computational methods can compliment experimental methods to expand our knowledge in outer membrane proteins. The main goal of this study is developing computational methods to gain a fundamental understanding of protein structure and function, and in turn using this knowledge to design outer membrane proteins. We first developed a computational approach to assess the folding free energy of outer membrane proteins by combining an empirical energy function with a reduced discrete state space. With this approach, we calculated depth-dependent transfer free energy scales of 20 amino acids, determined the native topology of proteins in the outer membrane, and assessed the effect of asymmetric bacterial outer membranes on the thermodynamic stability of proteins. We then developed a computation-guided strategy to design a novel outer membrane protein. A set of sequence motifs that may be important for the outer membrane protein organization was identified from combinatorial analysis. Strands that are involved in oligomerization have few such motifs, and were replaced with those that are enriched with motifs. Experimental validation proved the success of our design as the resulting protein OmpGF folded into predominant β-sheet structures, and formed a monomeric ion-conducting pore. We also discovered a general mechanism that drives oligomerization of outer membrane proteins. A sequence motif that is specific to trimeric porin family was identified, and was found to form inter-molecular interactions that are conserved in this family. Experimental examination of OmpF and PorB proteins proved that these inter-molecular interactions are essential for trimer formation. Overall, findings from this study provided additional understanding of folding principles of outer membrane proteins, and paved the road for designing novel outer membrane proteins.