Insertion in barnase of a loop sequence from ribonuclease T1. Investigating sequence and structure alignments by protein engineering.


Abstract

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

Submission Details

ID: XRkFygCh

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:34 p.m.

Version: 1

Publication Details
Vuilleumier S;Fersht AR,Eur. J. Biochem. (1994) Insertion in barnase of a loop sequence from ribonuclease T1. Investigating sequence and structure alignments by protein engineering. PMID:8181455
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1A2P 1998-01-07T00:00:00+0000 1.5 BARNASE WILDTYPE STRUCTURE AT 1.5 ANGSTROMS RESOLUTION
1B20 1998-12-03T00:00:00+0000 1.7 DELETION OF A BURIED SALT-BRIDGE IN BARNASE
1B21 1998-12-03T00:00:00+0000 2.0 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1B27 1998-12-04T00:00:00+0000 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2S 1998-11-30T00:00:00+0000 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2U 1998-12-01T00:00:00+0000 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1B2X 1998-12-03T00:00:00+0000 1.8 BARNASE WILDTYPE STRUCTURE AT PH 7.5 FROM A CRYO_COOLED CRYSTAL AT 100K
1B2Z 1998-12-03T00:00:00+0000 2.03 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1B3S 1998-12-01T00:00:00+0000 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BAN 1993-05-19T00:00:00+0000 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
97.3 Ribonuclease P35078 RN_BACCI
100.0 Ribonuclease P00648 RNBR_BACAM