NASA Astrobiology Institute (NAI)

1. Early Metabolic Pathways

   Project Investigators: Andrew Pohorille, Burckhard Seelig, Jack Szostak, Michael Wilson

   Other Project Members

   Chenyu Wei (Research Staff)

   Summary

   The project is aimed at characterizing the emergence of functional proteins and their early evolution leading to the formation of primitive metabolism in ancestors of contemporary cells. Through a combination of molecular biology and computer modeling we investigate the origins of both water-soluble enzymes and membrane proteins that mediate transport of small molecules and ions across cell walls.

   Astrobiology Roadmap Objectives:

   • Objective 3.2: Origins and evolution of functional biomolecules
   • Objective 3.4: Origins of cellularity and protobiological systems

   Project Progress

   In order to understand the evolutionary origins of functional macromolecules we continued studies on the first enzyme with novel function selected through in vitro evolution. This enzyme joins two fragments of RNA into a single strand (acts as an RNA ligase). Current studies concentrated on enhancing enzyme stability at higher temperature in order to obtain a more compact structure and increase enzymatic activity. A selection scheme, shown in Figure 1, was slightly modified to include incubation of the mRNA-displayed proteins at an elevated temperature of 65°C. The resulting pool of proteins was cloned and sequenced, and the sequences were analyzed. Four clones were assayed for ligation activity at 65°C. All of them showed such activity, whereas the ligases resulting from the original selection, assayed under the same conditions, yielded no detectable ligation product. All four heat-evolved ligases showed also an increased activity at room temperature compared to the original ligases. The results demonstrate the evolutionary potential of simple proteins to increase both their stability and enzymatic activity.

   
   

   General selection scheme for enzymes that catalyse bond-forming reactions. A DNA library is transcribed into RNA, cross-linked to a 39-puromycin oligonucleotide, and in vitro translated. The library of mRNA-displayed proteins is reverse transcribed with a primer bearing substrate A. Substrate B, which carries an anchor group, is added. Proteins that join A and B attach the anchor group to their encoding cDNA. Selected cDNA sequences are then amplified by Polymerase Chain Reaction (PCR), and used as input for the next round.

   In order to elucidate further the evolutionary potential of simple proteins we used computer design approach to redesign the previously evolved protein that binds adenosine triphosphate such that it can bind another, closely related molecule – guanosine triphosphate. This required extending one loop in this protein such that it could form sequence-dependent hydrogen bonds capable of favorable interactions with either adenine or guanine. One such hydrogen-bonding scheme is shown in Figure 2. These studies provide clues to identifying selection criteria in protobiological evolution because proteins lacking sufficient evolutionary flexibility to improve their catalytic efficiency or to alter their substrate specificity in response to a small number of mutations would have been eliminated through evolutionary pruning.

   
   

   Computer graphics representation of interactions between the guanosine triphosphate (on the right side) and amino acids side chains of a computer designed mutant protein capable of forming favorable, sequence-specific hydrogen bonds with guanine. Other mutants can form favorable hydrogen bonds with adenine. In the original protein, which contained a shorter loop, backbone atoms rather than side chains participated in hydrogen bonding. Therefore, modulating protein specificity through mutations was not possible.

Publications

Deamer, D. & Pohorille, A.  (2008).  Synthetic Cells and Life’s Origins.  Astrobiology, 8(2):453-455.

Pohorille, A.  (2008).  Chemical and Biological Determinants of Habitability.  Astrobiology, 8(2):327-330.

Pohorille, A., Wilson, M.A. & Wei, C.  (2007).  The Earliest Ion Channels.  Astrobiology, 7(3):492-493.

Pohorille, A., Wilson, M.A. & Wei, C.  (In press, 2008).  The Earliest Ion Channels.  In: K.M.a.W. Irvin (Ed.).  Proceedings of the Bioastronomy 2007.  ASP Conference Series.

Seelig, B. & Szostak, J.W.  (2007).  Selection and evolution of enzymes from a partially randomized non-catalytic scaffold.  Nature, 448(7155):828-831.