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
2KF5 2009-12-08 Barnase bound to d(CGAC), low pressure
2KF6 2009-12-08 Barnase bound to d(CGAC) high pressure
2KF3 2009-12-08 Barnase, low pressure reference NMR structure
2KF4 2009-12-08 Barnase high pressure structure
1FW7 2003-06-10 NMR STRUCTURE OF 15N-LABELED BARNASE
1BNR 1995-07-31 BARNASE
2C4B 2005-11-21 1.3 Inhibitor cystine knot protein McoEeTI fused to the catalytically inactive barnase mutant H102A
1A2P 1998-04-29 1.5 BARNASE WILDTYPE STRUCTURE AT 1.5 ANGSTROMS RESOLUTION
2ZA4 2008-05-20 1.58 Crystal Structural Analysis of Barnase-barstar Complex
1B20 1998-12-09 1.7 DELETION OF A BURIED SALT-BRIDGE IN BARNASE
1BRN 1994-01-31 1.76 SUBSITE BINDING IN AN RNASE: STRUCTURE OF A BARNASE-TETRANUCLEOTIDE COMPLEX AT 1.76 ANGSTROMS RESOLUTION
1B2X 1998-12-09 1.8 BARNASE WILDTYPE STRUCTURE AT PH 7.5 FROM A CRYO_COOLED CRYSTAL AT 100K
1B2S 1998-12-08 1.82 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1RNB 1992-07-15 1.9 CRYSTAL STRUCTURE OF A BARNASE-D(*GP*C) COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1X1Y 2005-04-26 1.9 Water-mediate interaction at aprotein-protein interface
1BRI 1995-07-10 1.9 BARNASE MUTANT WITH ILE 76 REPLACED BY ALA
3KCH 2010-03-09 1.94 Baranase crosslinked by glutaraldehyde
2F5M 2006-04-25 1.95 Cross-linked barnase soaked in bromo-ethanol
2F56 2006-04-25 1.96 Barnase cross-linked with glutaraldehyde soaked in 6M urea
1BSE 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRJ 1995-07-10 2.0 BARNASE MUTANT WITH ILE 88 REPLACED BY ALA
1B21 1998-12-09 2.0 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1BSA 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRS 1994-06-22 2.0 PROTEIN-PROTEIN RECOGNITION: CRYSTAL STRUCTURAL ANALYSIS OF A BARNASE-BARSTAR COMPLEX AT 2.0-A RESOLUTION
1BSC 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
2F5W 2006-04-25 2.0 Cross-linked barnase soaked in 3 M thiourea
1BNF 1995-07-10 2.0 BARNASE T70C/S92C DISULFIDE MUTANT
1BRK 1995-07-10 2.0 BARNASE MUTANT WITH ILE 96 REPLACED BY ALA
1BSB 1994-01-31 2.0 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1BRH 1995-07-10 2.0 BARNASE MUTANT WITH LEU 14 REPLACED BY ALA
1B2Z 1998-12-09 2.03 DELETION OF A BURIED SALT BRIDGE IN BARNASE
1BNS 1994-06-22 2.05 STRUCTURAL STUDIES OF BARNASE MUTANTS
1B2U 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1X1W 2005-04-26 2.1 Water-mediate interaction at aprotein-protein interface
1BNE 1995-07-10 2.1 BARNASE A43C/S80C DISULFIDE MUTANT
1BNG 1995-07-10 2.1 BARNASE S85C/H102C DISULFIDE MUTANT
1BNJ 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 9.0
1BNI 1995-09-15 2.1 BARNASE WILDTYPE STRUCTURE AT PH 6.0
1B27 1998-12-09 2.1 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
2F4Y 2006-04-25 2.15 Barnase cross-linked with glutaraldehyde
3Q3F 2012-01-25 2.17 Engineering Domain-Swapped Binding Interfaces by Mutually Exclusive Folding: Insertion of Ubiquitin into position 103 of Barnase
1YVS 1999-02-02 2.2 Trimeric domain swapped barnase
1BAN 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
1BAO 1993-10-31 2.2 THE CONTRIBUTION OF BURIED HYDROGEN BONDS TO PROTEIN STABILITY: THE CRYSTAL STRUCTURES OF TWO BARNASE MUTANTS
1BRG 1994-06-22 2.2 CRYSTALLOGRAPHIC ANALYSIS OF PHE->LEU SUBSTITUTION IN THE HYDROPHOBIC CORE OF BARNASE
3DA7 2009-04-14 2.25 A conformationally strained, circular permutant of barnase
1BSD 1994-01-31 2.3 CRYSTAL STRUCTURAL ANALYSIS OF MUTATIONS IN THE HYDROPHOBIC CORES OF BARNASE
1X1X 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1X1U 2005-04-26 2.3 Water-mediate interaction at aprotein-protein interface
1B3S 1998-12-09 2.39 STRUCTURAL RESPONSE TO MUTATION AT A PROTEIN-PROTEIN INTERFACE
1BGS 1994-04-30 2.6 RECOGNITION BETWEEN A BACTERIAL RIBONUCLEASE, BARNASE, AND ITS NATURAL INHIBITOR, BARSTAR

Relevant UniProtKB Entries

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