Research Projects

How life emerged on the earth is one of the most critical questions in biology. Proteins are exciting as they are the ultimate biomolecules created through trial and error in ~4 billion years and drive almost all life phenomena on earth. While protein science has made remarkable progress, the fundamental question of how proteins were born has not been completely resolved. Earth organisms have molecular systems centered around proteins, as one major advantage of using proteins over other polymers is sequence diversity. For example, since the number of atoms in the universe is estimated to be 10^80, it can be said that even a protein with only 100 residues has an amazingly vast sequence space. However, it is well-known that very few molecules function in this extensive combination. How, then, have organisms acquired functional proteins from diverse sequences? With probabilistic thinking, finding functional sequences in this nearly infinite sequence space is almost impossible. However, from an evolutionary point of view, the ancient proteins were considered simpler, which depended mainly on the "type of amino acid" and "length of peptide". Modern proteins/enzymes with large and complex structures are thought to have evolved from small and simple ancient proteins with "prototype-folds" (e.g., Rossmann-fold, ferredoxin-fold, and (beta/alpha)8-barrel). These prototype folds played essential roles in the early evolution of life, as they are often conserved in fundamental biochemical pathways. Specifically, the β-barrels are one of the commonly found structures in proteins/enzymes of diverse functionalities, of which a famously known one is the double-psi β-barrel (DPBB). The DPBB structure is part of key molecular machines having therapeutic and translational relevance. The DPBB core is conserved in (i) DNA and RNA polymerases-part of the gene expression system, (ii) Aspartate decarboxylase, Phosphotransacylases, Thymidine phosphorylase-part of metabolic pathways, and (iii) Valosin-containing protein (VCP), β-secretase-part of protein modification. However, how such proteins emerged on earth and acquired their functions remains elusive. To probe whether such prototype fold-embedded proteins can be born and made with simpler structures, here we propose a comprehensive investigation to resurrect the ancient core DPBB structure of VCP. By employing high-throughput computational protein design (CPD) strategies involving negative design, ensemble backbone design, ancestral sequence reconstruction, and structural scaffold-grafting, followed by experimental techniques (in collaboration), the functionally relevant DPBB cores of VCP will be resurrected. The sequence-structure-folding-stability relationships and catalytic functionalities of these resurrected proteins will be examined. This project will help bridge the gap in the evolutionary-functional relationship of macromolecular machines and have a substantial impact on therapeutics and bioengineering.