Evolutionäre Verwandtschaft zweier elementarer Protein-Faltungen: die (betaalpha)8-barrel und die (betaalpha)5- flavodoxin-ähnliche Faltung
Final Report Abstract
Nature displays a vast structural and functional diversity in contemporary proteins and it is of great interest how these protein structures and functions evolved. Molecular evolution studies have led to important insights to protein folding, function and design. Here we performed a sequence-based exploration of the structure space between two ancient and highly populated protein folds, the (βα)8- barrel and the flavodoxin-like fold. Using state-of-the-art tools for homology detection we found evidence of homology between proteins of both superfolds and furthermore detected a family of sequences that show intermediate features. We determined a representative crystal structure of one of its members, providing insights into the evolutionary link between two of the earliest protein folds. The comparison of the two folds was further tested in a rational design approach. Since protein folds are believed to have evolved by recombination from smaller but intrinsically stable peptide fragments, we applied this strategy in a design experiment. We combined structurally similar parts of a (βα)8- barrel and a flavodoxin-like protein creating a new hybrid protein. Upon solution of its crystal structure it became apparent that the new interface was not optimal. Thus, we applied computational design to optimize the interface between the fragments with five targeted mutations. This yielded a stable, monomeric protein whose predicted structure was verified by NMR spectroscopy. We then tested binding of a phosphorylated compound and detected that some affinity was present due to an intact phosphate-binding site provided by one parental fragment. With two additional mutations the affinity could be quickly improved to the level of natural proteins. The study illustrates the potential of recombining protein fragments with unique properties to design new and functional proteins, offering both a possible pathway for protein evolution and a protocol to rapidly engineer proteins for new applications.
Publications
- (2012) A highly stable protein chimera built from fragments of different folds. Protein Eng Des Sel; 25, 699-703
Shanmugaratnam S, Eisenbeis S & Höcker B
(See online at https://doi.org/10.1093/protein/gzs074) - (2012) The potential of fragment recombination for the rational design of proteins. J Am Chem Soc; 134, 4019- 22
Eisenbeis S, Proffitt W, Coles M, Truffault V, Shanmugaratnam S, Meiler J, Höcker B
(See online at https://doi.org/10.1021/ja211657k) - (2013) Engineering chimaeric proteins from fold fragments: “hopeful monsters” in protein design. Biochem Soc Trans; 41, 1137-40
Höcker B
(See online at https://doi.org/10.1042/BST20130099) - Design of chimeric proteins by combination of subdomain-sized fragments. Chapter 19. In Methods in Enzymology: Protein design. Ed. AE Keating. Elsevier 2013. Vol 523, page 389-405
Farias J & Höcker B
- (2014) Design of proteins from smaller fragments. Curr Opin in Struc Biol; 27C, 56-62
Höcker B
(See online at https://doi.org/10.1016/j.sbi.2014.04.007) - (2014) Evolutionary relationship of two ancient superfolds. Nature Chemical Biology; 10(9), 710-5
Farias-Rico JA, Schmidt S & Höcker B
(See online at https://doi.org/10.1038/NCHEMBIO.1579)
