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. 2011 Jan 21;47(3):973-5.
doi: 10.1039/c0cc03864d. Epub 2010 Nov 16.

Ribonuclease S Redux

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Free PMC article

Ribonuclease S Redux

Rex W Watkins et al. Chem Commun (Camb). .
Free PMC article

Abstract

The S-peptide and S-protein components of bovine pancreatic ribonuclease form a noncovalent complex with restored ribonucleolytic activity. Although this archetypal protein-fragment complementation system has been the object of historic work in protein chemistry, intrinsic limitations compromise its utility. Modern methods are shown to overcome those limitations and enable new applications.

Figures

Fig. 1
Fig. 1
Notional structure of “RNase–S”, the mixed disulfide of A4C S15 (red) and V118C S-protein (gray). Disulfide bonds (yellow) form between the indicated cysteine residues. The image is based on the known structure of the noncovalent S-protein·S15 complex.
Fig. 2
Fig. 2
Analysis of the proteolytic digestion of ribonucleases with SDS–PAGE. Left, products of the cleavage of wild-type RNase A by subtilisin. Right, products of the cleavage of DDDDK RNase A and wild-type RNase A by enterokinase.
Fig. 3
Fig. 3
Separation of S-peptide and S-protein components from DDDDK RNase A by reversed-phase HPLC.
Fig. 4
Fig. 4
Analysis of RNase–S semisynthesis with SDS–PAGE. Lane 1, RNase A; lane 2, RNase S; lane 3, S-protein derived from H12A/DDDDK/V118C RNase A; lane 4, A4C S-peptide + V118C S-protein of lane 3.
Fig. 5
Fig. 5
Analysis of RNase–S semisynthesis with zymogram electrophoresis. Lane 1, RNase A; lane 2, S-protein derived from H12A/DDDDK/V118C RNase A; lane 3, S15 + V118C S-protein of lane 2; lane 4, A4C S15 + V118C S-protein of lane 2; lane 5, RNase A; lane 6, commercial RNase S.
Fig. 6
Fig. 6
Initial velocities of RNA cleavage at high (0.15 µM) and low (25 pM) ribonuclease concentrations, relative to RNase A. “RNase S” refers to the noncovalent complex of S15 and the S-protein derived from H12A/DDDDK/V118 RNase A.

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