Functional assembly of a randomly cleaved protein.

Abstract

The sequence of a 939-amino acid polypeptide that is a member of the aminoacyl-tRNA synthetase class of enzymes has been aligned with sequences of 15 related proteins. This alignment guided the design of 18 fragment pairs that were tested for internal sequence complementarity by reconstitution of enzyme activity. Reconstitution was achieved with fragments that divide the protein at both nonconserved and conserved sequences, including locations proximal to or within elements believed to form critical elements of secondary structure. Structure assembly is sufficiently flexible to accommodate fusion of short segments of unrelated sequences at fragment junctions. Complementary chain packing interactions and chain flexibility appear to be widely distributed throughout the sequence and are sufficient to reconstruct large threedimensional structures from an array of disconnected pieces. The results may have implications for the evolution and assembly of large proteins.

Reconstitution of the enzyme AroA and its glyphosate tolerance by fragment complementation

“…This finding is consistent with previous observations [11,19]. However, splitting the enzyme in poorly conserved region does not always result in functional complementation.…”

“…Proteins reconstituted from fragments produced by limited proteolytic cleavage or genetic methods have been studied under both in vivo and in vitro conditions [9][10][11][12][13]. Successful reconstitution implies that the internal complementarity must be highly self-selective to overcome competing interactions with other protein sequences [14].…”

The appearance of pyrrolysine in tRNA His guanylyltransferase by neutral evolution

“…3B). As know from other studies (26) there are dispensable regions in proteins that can be deleted, yet the protein fragments can be functional in trans.…”

Identification of the Minimal Intracellular Vacuolating Domain of the Helicobacter pylori Vacuolating Toxin

“…It is possible that a putative VacA catalytic domain may be composed of noncontiguous domains requiring assembly before elaborating the catalytic activity of the enzyme. Although some enzymes can be dissected and the fragments functionally reassembled (23,19,49), this has not previously been demonstrated for any toxin A-fragment. Alternatively, p33 and p70 could conceivably possess independent functions, both of which are required for mediating an undefined mechanism to induce formation of vacuoles within the host cell cytosol.…”