Group I intron encoded proteins represent a novel class of site specific double strand endonucleases. The endonuclease activity of this class of proteins has been first demonstrated in vivo for I-Sce I which is encoded by a mitochondrial intron of Saccharomyces cerevisiae. Assays using crude cell extracts have shown that I-Sce I can be used in vitro as a restriction endonuclease potentially useful for recombinant DNA technology owing to its large recognition sequence (18 nucleotides). We report here the purification and the first detailed analysis of the in vitro activity and properties of I-Sce I.

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We studied the effects of n-propanol and pH on the structure of the apolipoprotein E3 N-terminal receptor binding domain, apo E3(1±191), to determine whether conditions similar to those occurring near lipid surfaces (decreased dielectric constant and pH) can mimic lipid-induced conformational changes in apo E3. The addition of 30% n-propanol, at pH 7, induces a conformational change in apo E3(1±191) as shown by changes in the intrinsic tryptophan fluorescence and by an increase in the Stokes radius of the majority of the protein from 3.0 to 4.1 nm, although the protein remains monomeric as shown by chemical cross-linking. These changes are accompanied by increased resistance to limited proteolysis with trypsin, chymotrypsin, subtilisin and endoproteinase glu-C, as is the case for apo E3(1±191) reconstituted into phospholipid/cholesterol lipid bicelles. Far and near UV circular dichroism showed that n-propanol increases the amount of calculated a-helical structure (42±65%) and alters the tertiary structure of the protein although not as much as when apo E3(1±191) is incorporated into lipid bicelles. In the absence of n-propanol, lowering the pH to 4.5 decreases the Stokes radius of the majority of the protein somewhat, with little effect upon the secondary and the tertiary structures. The addition of 30% n-propanol at pH 4.5 increases the Stokes radius of apo E3(1±191) from 2.2 to 5.0 nm, even more than at pH 7 (3.0±4.1 nm) although the protein still remains predominantly monomeric. There is increased resistance to limited proteolysis with endoproteinase glu-C. As assessed by far and near UV circular dichroism, the addition of 30% n-propanol at pH 4.5, in contrast to pH 7, markedly increases the a-helical structure and changes the tertiary structure of the protein similarly to that resulting from the incorporation of apo E3(1±191) into lipid bicelles. The results suggest that a combination of n-propanol and low pH in aqueous solutions may be useful as a simple model system for studying conformational changes in apo E3 similar to those, which occur upon interaction of the protein with lipids. [7]. Mutations in apo E which destroy interactions with the low density lipoprotein (LDL) receptor are an underlying cause of Type III hyperlipoproteinemia [8] and the protein also has been associated with the development of Alzheimer's disease [6]. The activity of apo E in these functions depends upon its precise three dimensional structure which, in turn, depends upon both the sequence and the environment of the protein. For example, the mutation giving rise to the apo E2 isoform disrupts a naturally occurring salt bridge [9] and permits the formation of a new salt bridge [10] thus altering a portion of the structure of the molecule which appears to be critical for receptor binding [11]. The environment also has an effect upon the structure as illustrated by the fact that the formation of this new salt bridge, which inactivates apo E receptor binding, appears to be variable and to depend upon the type and lipid composition...

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