Insertion in barnase of a loop sequence from ribonuclease T1. Investigating sequence and structure alignments by protein engineering.
Barnase was mutated by inserting into its active site loop sequences found in the related enzyme ribonuclease T1 (RNase T1), according to either structural or sequential similarity alignments. The barnase/RNase T1 hybrid corresponding to the structural alignment of the two proteins, endo-[RNaseT1-(93-99)]102abarnase, contains RNase T1 residues at positions 93-99 inserted between residues at positions 102 and 103 of barnase. The other constructed mutant, endo-[RNaseT1-(95-98)]104abarnase, has RNase T1 residues at positions 95-98 inserted between residues at positions 104 and 105 in barnase, corresponding to published sequence alignments of the two proteins in this region. The mutants were characterized by absorbance, fluorescence and CD spectroscopy; the stability, folding and unfolding kinetics, and catalytic activity were measured and compared with the wild-type enzyme. Endo-[RNaseT1-(93-99)]102abarnase, the mutant protein corresponding to the structural alignment of barnase with ribonuclease T1, shows a slightly higher stability (approximately 5 kJ/mol) towards urea and heat denaturation than the mutant endo-[RNaseT1-(95-98)]104abarnase, designed according to a sequence alignment between the two enzymes. Both mutants have very low catalytic activity, although the effect of mutation is almost entirely limited to kcat in the case of the mutant corresponding to the structural alignment between barnase and ribonuclease T1, while both kcat and Km are affected in the mutant corresponding to the sequence alignment between the two enzymes. Thus, the superiority of structural over sequential alignments cannot be supported conclusively by direct experiment in the present case.
Vuilleumier
S
S
Cambridge Centre for Protein Engineering, England.
Fersht
A R
AR
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002384:Catalysis; D002942:Circular Dichroism; D004926:Escherichia coli / Q000201:enzymology; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D006163:Ribonuclease T1 / Q000737:chemistry / Q000235:genetics*; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000378:metabolism
D001426:Bacterial Proteins; D014508:Urea; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease; D006163:Ribonuclease T1