Dissection of an enzyme by protein engineering. The N and C-terminal fragments of barnase form a native-like complex with restored enzymic activity.
A method is described for producing fragments of a protein suitable for studies of protein folding. The codon for a single methionine residue is introduced into the cloned gene of barnase, and the gene product cleaved with cyanogen bromide. The site of mutation was chosen to be at the surface of the protein in a region connecting segments of secondary structure in the native enzyme. The alpha + beta protein was mutated from Val36----Met, and split into two fragments, B(1-36) containing the alpha-helical regions and B(37-110), the beta-sheet. The fragments were purified by ion exchange chromatography. Neither retains catalytic activity. Fluorescence, circular dichroism, and 1H nuclear magnetic resonance data indicate that their structures are each close to that of random-coil peptides. The two fragments associate to form a tight complex (Kd = 0.2 to 0.6 microM), which displays spectroscopic properties similar to those of the uncleaved protein. The catalytic activity is restored in the complex with a value for Km similar to that for native enzyme but with kcat reduced about three- to fourfold. The second-order rate constant for association on mixing fragments in the concentration range 2.5 to 7.5 microM is 1 x 10(5) s-1 M-1.
Sancho
J
J
Department of Chemistry, University of Cambridge, U.K.
Fersht
A R
AR
D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D002942:Circular Dichroism; D004789:Enzyme Activation; D006868:Hydrolysis; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D015202:Protein Engineering; D012260:Ribonucleases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence
D001426:Bacterial Proteins; D010446:Peptide Fragments; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease