Exon-coded polypeptides as primordial enzymes.
Abstract: Abstract Proteins are diverse and very complex molecules capable of
delivering enormous rate accelerations over uncatalized reactions. Questions regarding
origin and evolution of protein activity only recently started to receive experimental
answers. Two theories have been put forward that propose possible mechanisms for the origin
of protein activity: "The Origin of Genes" by Gilber... read moret and "Exons as Microgenes" by Knowles.
Both theories indicate the crucial role of exon shuffling in generation of primordial genes
coding for first proteins. However, Knowles suggests an additional statement that each
exon-coded polypeptide was independently translated and primordial enzymes were
non-covalent assemblies of these protein fragments. In our study we aimed at providing
experimental evidence to support "Exons as Microgenes theory". Two fusion enzymes were
generated where flexible linker was incorporated at the position defined by exon/exon
boundaries of human adenylate kinase 1 (AK1). The length of this linker was such that
allowed for sufficient spatial separation of protein fragments thus preserving their
identities as individual protein fragments. The crucial importance of the exon/exon
boundary for introduction of linker was demonstrated on the example of control fusion
proteins, where the linker was moved away from the exon/exon boundary by a few amino acids.
Enzymatically competent reassemblies were only observed in the case of fusion proteins with
linkers situated at exon/exon boundaries. This correlation was demonstrated to be relevant
both in vitro and in vivo. Relative to control wild type enzyme, specific activities were
only moderately lower, 2 and 9 times for fusion proteins containing one linker and 2
linkers (at two different exon/exon boundaries) respectively. Designed protein constructs
were structurally characterized via circular dichroism (CD) analysis. All protein
constructs retained folding patterns similar to that of control full-length enzyme, though
their folding was found to be diminished. Binding constants of both substrates were found
to be higher than those of wild type enzyme, which could account for observed lower
activity of fusion proteins. The broader utility of the linker mediated exon-coded
polypeptide reassembly approach is yet to be shown on other proteins.
Thesis (Ph.D.)--Tufts University, 2012.
Submitted to the Dept. of Chemistry.
Advisor: Krishna Kumar.
Committee: David Walt, James Baleja, and David Casebier.
Keywords: Chemistry, and Biochemistry.read less