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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.

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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.

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