Isolation and physical characterization of random insertions in Staphylococcal nuclease.
Using genetic engineering techniques we generated randomly located internal tandem duplications of random size within Staphylococcal nuclease. Those insertions, possessing greater than 0.1% of normal activity, were sequenced and characterized physically. Insertions were found to begin and end in regions possessing secondary structure as well as in regions without secondary structure. All proteins remained folded and monomeric, although one mutant appeared, by both circular dichroism and size exclusion chromatography, to be partially unfolded. The stability of the insertions as assayed by guanidine hydrochloride denaturation ranged from nearly normal to destabilized by almost 4 kcal per mol. The activities of the insertion mutants ranged from 1/30 to 1/2000 of the parental nuclease.
Nguyen
D M
DM
Department of Biology, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD, 21218, USA.
Schleif
R F
RF
D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D005818:Genetic Engineering; D019791:Guanidine; D007700:Kinetics; D008836:Micrococcal Nuclease / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008958:Models, Molecular; D016254:Mutagenesis, Insertional; D009154:Mutation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013210:Staphylococcus / Q000201:enzymology* / Q000235:genetics; D020080:Tandem Repeat Sequences / Q000235:genetics*; D013816:Thermodynamics; D014169:Transformation, Bacterial
D011994:Recombinant Proteins; D008836:Micrococcal Nuclease; D019791:Guanidine