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. Study holds ProTherm entries: 7652, 7653, 7654 Extra Details: active site loop sequence; structural or sequential similarity;,hybrid; catalytic activity; sequence alignment
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||7|
|Proteins||Ribonuclease ; Ribonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dG ; Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O|
|Libraries||Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR|