Book of Abstracts: Albany 2009
June 16-20 2009
© Adenine Press (2008)
Catalytic Partnership Between Inteins and Their Extein Neighbors
Here we describe the development and use of a FRET-based reporter assay for tracking intein activity in vitro and in living cells. As shown below, the native ?extein? substrates of a self-splicing intein were replaced with the naturally FRET-active cyan and yellow fluorescent proteins. Native extein residues near the splice junction that might influence intein activity were maintained. In vitro and in vivo analysis of the resulting protein showed high FRET signal associated with the spliced product and the unspliced precursor. Low FRET was affiliated with the products of N-extein cleavage.
We have used this FRET-active intein to test the hypothesis that non-reacting extein residues influence protein splicing. Extein residues that perturb the stability of high-energy intermediates formed during splicing were searched for and identified in mutant expression libraries on the basis of deviant in vivo FRET readings. Once selected by in vivo screening and cell sorting, variants were characterized by an analogous FRET-based assay that allowed for continuous, parallel, kinetic monitoring of intein activity in crude cell extracts.
Results of this screen indicate that mutations in non-reacting extein residues can have pronounced effects on the stability of splicing intermediates, with consequent changes in the yield of spliced product and the rate at which it is formed. These observations seem to contrast with the generally held notion that an intein can excise itself from ?virtually anywhere? within a host extein sequence. The existence of these extein effects and their magnitude further implies that intein integration sites may be selected, both in nature and in the biotechnological uses of inteins, in a manner more judicious than presently appreciated.