NASA Astrobiology Institute (NAI)

1. Early Metabolic Pathways

   Project Investigators: Andrew Pohorille

   Other Project Members

   John Chaput (Research Staff)

   Burckhard Seelig (Postdoc)

   Jack Szostak (Co-Investigator)

   Michael Wilson (Co-Investigator)

   Sharef Mansy (Postdoc)

   Chenyu Wei (Research Staff)

   Astrobiology Roadmap Objectives:

   • Objective 3: Replicating, catalytic systems capable of evolution, and construct laboratory models of metabolism in primitive living systems.
   • Objective 3: Replicating, catalytic systems capable of evolution, and construct laboratory models of metabolism in primitive living systems.

   Project Progress

   We continue to employ both experimental and computational approaches to investigate the evolutionary origins of functional macromolecules. We conducted the first laboratory evolution of a completely new non-biological enzyme that joins two fragments of RNA into a single strand (it acts as an RNA ligase). The enzyme was evolved from a partially randomized non-catalytic scaffold protein. NMR spectroscopy of the purified enzyme shows that most of the protein is well structured. The nature of amino acid substitutions during in vitro evolution indicates that the initial protein structure underwent at least local refolding. The results demonstrate that novel functions (and possibly different structures) can be obtained through a limited number of mutations in sequences of small proteins that might serve as models for ancestral macromolecules. Nature magazine has accepted the manuscript describing this work.

   We continued to explore the evolutionary optimization of our previously evolved ATP-binding protein. NMR and X-ray crystallographic studies have revealed an unexpectedly strong role of surface residue interactions in stabilizing this small protein. The results help us to elucidate pathways by which primordial protein sequences attain increased degrees of functionality through the systematic accumulation of point mutations. (2 papers, one in PLoS ONE, and one in press in J. Mol. Biol.)

   To explain how primordial proteins could have performed an essential cellular function of transporting ions across cell walls, which is carried out by some of the most complex protein assemblies of modern cells, we studied the antiamoebin channel using molecular dynamics computer simulations. This channel consists simply of 8 identical helices, each 16 amino acids in length. It achieves an efficiency that is comparable to that of a highly evolved voltage-gated potassium channel. On the basis of our results, we propose that channels evolved further towards high structural complexity because they needed to acquire mechanisms for precise regulation rather than to improve efficiency. Further, the observed dependence of amino acid sequence and function of membrane proteins on the nature of membrane-forming material suggests that channels and membranes might have co-evolved.

   
   Figure 1. A top view of the antiamoebin octameric channel. The amino acid sequence of antiamoebin is Phe-Aib-Aib-Iva-Gly-Leu-Aib-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phol, where Aib is α-aminoisobutiric acid, Iva is isovaline and Hyp is hydroproline Ions are transported across membrane bilayers through the pore in the middle.

   
   Figure 2. The cross-section of the antiamoebin channel spanning the phospholipids bilayer (dark green and gray) surrounded by 0.5 M aqueous solution of KCl. Potassium ions are yellow and chloride ions are blue. Note two potassium ions inside the channel.

   
   Figure 3. Sequences of the starting library and selected ligases (from Seelig & Szostak, Nature (2007), in press). Loop regions are highlighted in light blue. The cysteines highlighted in orange constitute the two pairs of CXnC (n = 2 or 5) motifs that coordinate zinc ions in the original hRXR domain. Randomized amino acids in the library are shown as x. Dashes indicate amino acids that are the same as in the starting library, whereas periods highlighted in grey symbolize deletions.

Publications

Mansy, S.S., Zhang, J., Kummerle, R., Nilsson, M., Chou, J.J., Szostak, J.W. & Chaput, J.C.  (In Press, 2007).  Structure and Evolutionary Analysis of a Non-biological ATP-binding Protein.  J. Mol. Biol..

Pohorille, A.  (2007).  Unanswered questions in the origin of life.  Biology: the Dynamic Science.

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

Seelig, B. & Szostak, J.W.  (In Press, 2007).  Selection and Evolution of Enzymes from a Partially Randomized Non-Catalytic Scaffold.  Nature.

Smith, M.D., Rosenow, M.A., Wang, M., Allen, J.P., Szostak, J.W. & Chaput, J.C.  (2007).  Structural Insights into the Evolution of a Non-Biological Protein: Importance of Surface Residues in Protein Fold Optimization.  PLoS One, 2(5):e467.