2014 Annual Science Report
NASA Ames Research Center Reporting | SEP 2013 – DEC 2014
Origins of Functional Proteins and the Early Evolution of Metabolism
The main goal of this project is to identify critical requirements for the emergence of biological complexity in early habitable environments by examining key steps in the origins and early evolution of functional proteins and metabolic reaction networks. Applying a combination of experimental and theoretical methods, we investigate whether protein functionality can arise from an inventory of polypeptides that might have naturally existed in habitable environments, investigate how primordial proteins could evolve through the diversification of their structure and function and determine how simple proteins could carry out seemingly complex functions that are essential to life. This work offers unique information about the earliest evolution of cellular systems that has not been available from other studies.
We continued investigations of model primordial enzymes. One approach to creating such enzymes is to introduce mutations into an existing protein to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques such as directed evolution, is should be ensured that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that isolated enzymes are both stable and have the desired catalytic properties. We use the in vitro selection technique mRNA display to achieve both in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65 °C. The most efficient enzyme, ligase 10C, was not only active at 65 °C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. (insert Fig. 1) Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. These results highlight the versatility of the in vitro selection technique mRNA display as a powerful method for the isolation of thermostable models of primordial enzymes.
Another essential function of primordial proteins was transmembrane proton transfer that facilitated transduction of energy for metabolic functions. The reliable, efficient and controlled generation of proton gradients became possible only with the emergence of active proton pumps. To understand this process, we studied a small, hexameric, transmembrane M2 proton channel from influenza A virus as a model for proton transport. Remarkably, the M2 channel and its truncated version built of only 24 residues per monomer contain almost all mechanistic features of markedly larger, modern proton pumps. Nδ in the imidazole rings of the His37 sensor can be considered as the primary acceptor that receives a proton from the aqueous cavity near Gly34. In response to protonation, histidine undergoes conformational change shuttling the proton to the opposite side of the sensor. Deprotonation on the outgoing side is followed by the return of histidine to its initial conformation preventing back transfer of the proton to the cavity. Trp41 and Asp44 also ensure directional proton transfer to the aqueous solution on the outgoing side. The Val27 gate protects protons that enter the cavity from returning to the incoming side. It appears that the only missing element that would turn M2 to a proton pump is a proton source that would be located near the aqueous cavity, as illustrated in Fig. 2. Example of such proton source might be polycyclic aromatic hydrocarbon (PAH) molecules that are sensitive to ultraviolet lights. PAH are believed to be abundant on the early earth and have been proposed to provide protons for protocellular functions. The smallest PAH, naphthalene, is comparable in size to amantadine, a drug known to reside in the Gly34 cavity. Although M2 is not an evolutionarily early protein, the analysis of its structure and mechanism of action leads to a conclusion that a design that captures the main features of proton pumps could also apply to simple proteins that might have existed early in the protobiological era. Such proteins might have been at the roots of active proton transport.
PROJECT MEMBERS:Andrew Pohorille
RELATED OBJECTIVES:Objective 3.2
Origins and evolution of functional biomolecules
Origins of cellularity and protobiological systems