Structural stability and endonuclease activity of a PI-SceI GFP-fusion protein.
Homing endonucleases are site-specific and rare cutting endonucleases often encoded by intron or intein containing genes. They lead to the rapid spread of the genetic element that hosts them by a process termed 'homing'; and ultimately the allele containing the element will be fixed in the population. PI-SceI, an endonuclease encoded as a protein insert or intein within the yeast V-ATPase catalytic subunit encoding gene (vma1), is among the best characterized homing endonucleases. The structures of the Sce VMA1 intein and of the intein bound to its target site are known. Extensive biochemical studies performed on the PI-SceI enzyme provide information useful to recognize critical amino acids involved in self-splicing and endonuclease functions of the protein. Here we describe an insertion of the Green Fluorescence Protein (GFP) into a loop which is located between the endonuclease and splicing domains of the Sce VMA1 intein. The GFP is functional and the additional GFP domain does not prevent intein excision and endonuclease activity. However, the endonuclease activity of the newly engineered protein was different from the wild-type protein in that it required the presence of Mn(2+) and not Mg(2+) metal cations for activity.
Senejani
Alireza G
AG
Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA.
Gogarten
J Peter
JP
D001665:Binding Sites; D020134:Catalytic Domain; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D047668:Inteins; D008345:Manganese / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485192:green fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins; D008345:Manganese; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific; D006180:Proton-Translocating ATPases; C081687:VMA1 protein, S cerevisiae