Nuclear Magnetic Resonance and Circular Dichroism Spectroscopy to study the folding state of artificial, primordial-like proteins

Abstract

Proteins are the primary molecular machinery for all known life. All proteins are made from the standard 20 amino acids in accordance to the universal genetic code of life; however, prior to the last universal common ancestor, this code was not set in stone and had to evolve to include each of the amino acids. The Seelig lab has previously generated libraries of random proteins using reduced sets of amino acids in order to simulate possible early protein functionality. The reduced sets of amino acids were based on the consensus chronology of incorporation into the genetic code and consisted of 5, 9, 16, and 20 of the amino acids used in modern proteins. This research aimed to characterize the structures of ATP binding proteins from each mRNA display library, primarily focusing on the 5AA and 9AA alphabet libraries. HSQC NMR and CD spectroscopy were used to characterize the structural and folding state of these proteins. Measurements from these spectroscopy experiments reveal secondary structure characteristics of up to 45% alpha helicity for individual variants. Further characterization efforts for these proteins are ongoing, including additional NMR and CD experiments as well as attempts to crystallize the proteins for X-ray crystallography.