Book of Abstracts: Albany 2005
Molecular Insights into Intein Structure and Function
Inteins are self-splicing protein elements that are of interest from mechanistic and biotechnological standpoints. Many naturally occurring inteins consist of two functionally independent domains, a protein splicing domain and an endonuclease domain. Previously, we generated a 168-residue mini-intein by removing the central endonuclease domain of the 440-residue Mtu recA intein (1). In addition, directed evolution experiments identified a mutation, V67L, that significantly improved the activity of the mini-intein (2). We have developed mini-inteins as linkers in fusion proteins for bioseparations (2, 3, 4). These mini-inteins have been successfully applied to single-step protein purification in laboratory-scale column mode, and the process has been scaled down more than 100-fold for fluidic separation on a rotating disk (5).
To gain molecular insight into the function of these mini-inteins, and to design even smaller derivatives, we have initiated structural studies. We have determined the three-dimensional structure of one mini-intein by MAD phasing methods using a Hg-derivative. The structure has been refined at 1.7 Å resolution. The overall fold closely resembles those of other mini-inteins, although this represents the first structure of the free splicing product and of a mini-intein that is not compromised by mutations of residues involved in the splicing mechanism.
Guided by the structural information, a number of derivatives (135-152 residues, with V67 or L67) have been generated. All of the new intein derivatives are functional in splicing. In addition, the effect of the V67L mutation was observed to be universal for all mini-inteins longer than 137 amino acids. Experiments to assess the susceptibility of the inteins to proteolysis as a function of temperature indicate that the role of the mutation appears to be in structural stabilization of the mini-inteins. The new derivatives form the basis of the next generation of molecules to be used as separation agents and biosensors in a wide range of medical and biotechnological applications.
References and Footnotes