Reconstitution of Escherichia coli RNase HI from the N-fragment with high helicity and the C-fragment with a disordered structure.
The Escherichia coli RNase HI variant with the Lys86-->Ala mutation is purified in two forms, as nicked and intact proteins. The nicked K86A protein, in which the N-fragment (Met1-Lys87) and the C-fragment (Arg88-Val155) remain associated, is enzymatically active. These N- and C-fragments were isolated and examined for reassociation. These peptides did not associate to form the nicked K86A protein at pH 3.0 in the absence of salt, but were associated, with a yield of 30-80%, when the pH was raised to 5.5 or when salt was added. Measurements of the CD spectra show that the alpha-helices are partially formed in the N-fragment at pH 3.0 in the absence of salt and are almost fully formed either at pH 5.5 or at pH 3.0 in the presence of 0.15 M NaCl. In contrast, the C-fragment remains almost fully disordered under these conditions. The N-fragment with this high (native-like) helicity shows the characteristics of a molten globule with respect to the content of the secondary and tertiary structures, the ability to bind a fluorescent probe (1-anilinonaphthalene-8-sulfonic acid), and the behavior on the thermal transition. These results suggest that the N-fragment contains an initial folding site, probably the alpha I-helix, and the completion of the folding in this site provides a surface that facilitates the folding of the C-fragment. This folding process may represent that of the intact RNase HI molecule.
Kanaya
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E
Protein Engineering Research Institute, Osaka, Japan.
Kanaya
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S
D001665:Binding Sites; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D017354:Point Mutation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D016914:Ribonuclease H / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012965:Sodium Chloride
D010446:Peptide Fragments; D012965:Sodium Chloride; D016914:Ribonuclease H; C078856:ribonuclease HI