Spermine stabilization of folded ribonuclease T1.


Abstract

The interaction of ribonuclease T1 with tetraprotonated spermine (SPM4+), Mg2+, phosphate and other ionic ligands at pH 6.0 was investigated in binding experiments at 25 degrees C and/or by their effects on the midpoint temperature for thermal unfolding of the enzyme. SPM4+ binding with the native protein at 25 degrees C was characterized by an association constant of approximately 2 x 10(4) M-1. This ligand also binds to the unfolded protein but with a approximately 35-fold lower affinity. Phosphate binds at the active site whereas Mg2+ and SPM4+ cations compete for binding at a polyanionic locus that probably involves residues Glu-28, Asp-29, and Glu-31 at the C-terminal end of the alpha-helix. Steady-state kinetic studies using minimal RNA substrates demonstrated that SPM4+ binding with the enzyme does not affect its catalytic activity. SPM4+ also preferentially binds with the folded form of the disulfide-reduced enzyme which has the same or slightly enhanced catalytic properties compared with native ribonuclease T1. The unfolding rate for the native protein in 8 M urea was approximately 8-fold lower in the presence of 0.05 M SPM4+. SPM4+ appears to increase the amplitude of an unobserved fast phase(s) for refolding of the native enzyme. A single kinetic phase characterized refolding of the reduced enzyme which was slightly faster than the slowest refolding phase for the native form. Study holds ProTherm entries: 5173 Extra Details: ionic ligands; association constant; polyanionic locus;,alpha-helix; catalytic properties

Submission Details

ID: iBzycwwB

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Walz FG;Kitareewan S,J. Biol. Chem. (1990) Spermine stabilization of folded ribonuclease T1. PMID:1970567
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
1YGW 1997-10-08 NMR STRUCTURE OF RIBONUCLEASE T1, 34 STRUCTURES
1IYY 2003-10-07 NMR STRUCTURE OF Gln25-RIBONUCLEASE T1, 24 STRUCTURES
1I0V 2001-02-14 1.23 Ribonuclease T1 in complex with 2'GMP (form I crystal)
4ODK 2015-01-14 1.4 Structure of SlyD from Thermus thermophilus in complex with T1 peptide
9RNT 1993-01-15 1.5 RIBONUCLEASE T1 WITH FREE RECOGNITION AND CATALYTIC SITE: CRYSTAL STRUCTURE ANALYSIS AT 1.5 ANGSTROMS RESOLUTION
1LOY 2002-08-21 1.55 X-ray structure of the H40A/E58A mutant of Ribonuclease T1 complexed with 3'-guanosine monophosphate
1LOV 2002-08-21 1.55 X-ray structure of the E58A mutant of Ribonuclease T1 complexed with 3'-guanosine monophosphate
1I0X 2001-02-14 1.65 RIBONUCLEASE T1 IN COMPLEX WITH 2'GMP (FORM II CRYSTAL)
4GSP 1998-08-12 1.65 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 7 DAYS
1Q9E 2004-03-23 1.7 RNase T1 variant with adenine specificity
1HYF 2001-02-14 1.7 RIBONUCLEASE T1 V16A MUTANT IN COMPLEX WITH SR2+
4BIR 1998-07-15 1.7 RIBONUCLEASE T1: FREE HIS92GLN MUTANT
1RGA 1993-10-31 1.7 CRYSTAL STRUCTURE OF RNASE T1 WITH 3'-GMP AND GUANOSINE: A PRODUCT COMPLEX
1I3I 2001-03-07 1.76 Ribonuclease T1 V78T mutant
3BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
5BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
1I2E 2001-03-07 1.8 Ribonuclease T1 V16A mutant, form I
1FZU 2000-10-25 1.8 RNAse T1 V78A mutant
3RNT 1989-10-15 1.8 CRYSTAL STRUCTURE OF GUANOSINE-FREE RIBONUCLEASE T1, COMPLEXED WITH VANADATE(V), SUGGESTS CONFORMATIONAL CHANGE UPON SUBSTRATE BINDING
3BIR 1997-12-31 1.8 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 BY ASN AND GLN SUBSTITUTIONS
2GSP 1998-08-12 1.8 RIBONUCLEASE T1/2',3'-CGPS AND 3'-GMP, 2 DAYS
6RNT 1993-01-15 1.8 CRYSTAL STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ADENOSINE 2'-MONOPHOSPHATE AT 1.8-ANGSTROMS RESOLUTION
4BU4 1998-09-23 1.8 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
1BIR 1996-08-17 1.8 RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP
1RN4 1993-01-15 1.8 HIS92ALA MUTATION IN RIBONUCLEASE T1 INDUCES SEGMENTAL FLEXIBILITY. AN X-RAY STUDY
2AAE 1994-01-31 1.8 THE ROLE OF HISTIDINE-40 IN RIBONUCLEASE T1 CATALYSIS: THREE-DIMENSIONAL STRUCTURES OF THE PARTIALLY ACTIVE HIS40LYS MUTANT
5GSP 1998-03-18 1.8 RIBONUCLEASE T1/3'-GMP, 9 WEEKS
1HZ1 2001-01-31 1.8 RIBONUCLEASE T1 V16A MUTANT IN COMPLEX WITH MG2+
2RNT 1989-10-15 1.8 THREE-DIMENSIONAL STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH GUANYLYL-2(PRIME),5(PRIME)-GUANOSINE AT 1.8 ANGSTROMS RESOLUTION
1DET 1996-07-11 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
8RNT 1993-01-15 1.8 STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ZINC(II) AT 1.8 ANGSTROMS RESOLUTION: A ZN2+.6H2O.CARBOXYLATE CLATHRATE
1RN1 1994-01-31 1.84 THREE-DIMENSIONAL STRUCTURE OF GLN 25-RIBONUCLEASE T1 AT 1.84 ANGSTROMS RESOLUTION: STRUCTURAL VARIATIONS AT THE BASE RECOGNITION AND CATALYTIC SITES
1I2G 2001-03-07 1.85 Ribonuclease T1 V16T mutant
1G02 2000-10-25 1.86 Ribonuclease T1 V16S mutant
1RGK 1993-01-15 1.87 RNASE T1 MUTANT GLU46GLN BINDS THE INHIBITORS 2'GMP AND 2'AMP AT THE 3' SUBSITE
2HOH 1998-09-23 1.9 RIBONUCLEASE T1 (N9A MUTANT) COMPLEXED WITH 2'GMP
1RNT 1987-10-16 1.9 RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURE OF THE RIBONUCLEASE T1(ASTERISK)2(PRIME)-GUANYLIC ACID COMPLEX AT 1.9 ANGSTROMS RESOLUTION
1BU4 1999-02-16 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
1LOW 2002-08-21 1.9 X-ray structure of the H40A mutant of Ribonuclease T1 complexed with 3'-guanosine monophosphate
1RLS 1994-12-20 1.9 CRYSTAL STRUCTURE OF RNASE T1 COMPLEXED WITH THE PRODUCT NUCLEOTIDE 3'-GMP. STRUCTURAL EVIDENCE FOR DIRECT INTERACTION OF HISTIDINE 40 AND GLUTAMIC ACID 58 WITH THE 2'-HYDROXYL GROUP OF RIBOSE
7RNT 1993-01-15 1.9 CRYSTAL STRUCTURE OF THE TYR45TRP MUTANT OF RIBONUCLEASE T1 IN A COMPLEX WITH 2'-ADENYLIC ACID
1LRA 1994-01-31 1.9 CRYSTALLOGRAPHIC STUDY OF GLU 58 ALA RNASE T1(ASTERISK)2'-GUANOSINE MONOPHOSPHATE AT 1.9 ANGSTROMS RESOLUTION
1BVI 1998-09-23 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
3GSP 1998-08-12 1.9 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 4 DAYS
1I2F 2001-03-07 1.95 Ribonuclease T1 V16A mutant, form II
3HOH 1998-09-16 1.95 RIBONUCLEASE T1 (THR93GLN MUTANT) COMPLEXED WITH 2'GMP
2BU4 1998-09-23 1.95 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
3SYU 2012-03-28 1.95 Re-refined coordinates for pdb entry 1det - ribonuclease T1 carboxymethylated at GLU 58 in complex with 2'GMP
1RHL 1998-10-14 1.95 RIBONUCLEASE T1 COMPLEXED WITH 2'GMP/G23A MUTANT
2AAD 1994-01-31 2.0 THE ROLE OF HISTIDINE-40 IN RIBONUCLEASE T1 CATALYSIS: THREE-DIMENSIONAL STRUCTURES OF THE PARTIALLY ACTIVE HIS40LYS MUTANT
1B2M 1999-03-25 2.0 THREE-DIMENSIONAL STRUCTURE OF RIBONULCEASE T1 COMPLEXED WITH AN ISOSTERIC PHOSPHONATE ANALOGUE OF GPU: ALTERNATE SUBSTRATE BINDING MODES AND CATALYSIS.
5BIR 1997-12-31 2.0 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 USING ASN AND GLN MUTATIONS
1FYS 2000-10-25 2.0 Ribonuclease T1 V16C mutant
7GSP 1998-03-18 2.0 RIBONUCLEASE T1/2',3'-CGPS, NON-PRODUCTIVE
1RGC 1994-01-31 2.0 THE COMPLEX BETWEEN RIBONUCLEASE T1 AND 3'-GUANYLIC ACID SUGGESTS GEOMETRY OF ENZYMATIC REACTION PATH. AN X-RAY STUDY
1RGL 1993-01-15 2.0 RNASE T1 MUTANT GLU46GLN BINDS THE INHIBITORS 2'GMP AND 2'AMP AT THE 3' SUBSITE
5HOH 1998-09-23 2.0 RIBONUCLEASE T1 (ASN9ALA/THR93ALA DOUBLEMUTANT) COMPLEXED WITH 2'GMP
4HOH 1998-09-23 2.05 RIBONUCLEASE T1 (THR93ALA MUTANT) COMPLEXED WITH 2'GMP
1TTO 2005-09-06 2.1 Crystal structure of the Rnase T1 variant R2
4RNT 1992-01-15 2.2 HIS 92 ALA MUTATION IN RIBONUCLEASE T1 INDUCES SEGMENTAL FLEXIBILITY. AN X-RAY STUDY
6GSP 1998-03-18 2.2 RIBONUCLEASE T1/3'-GMP, 15 WEEKS
1GSP 1998-02-25 2.2 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS, 1 DAY
1CH0 1999-12-22 2.3 RNASE T1 VARIANT WITH ALTERED GUANINE BINDING SEGMENT
2BIR 1997-06-16 2.3 ADDITIVITY OF SUBSTRATE BINDING IN RIBONUCLEASE T1 (Y42A MUTANT)
1TRQ 1994-04-30 2.3 X-RAY CRYSTALLOGRAPHIC AND CALORIMERIC STUDIES OF THE EFFECTS OF THE MUTATION TRP 59 TYR IN RIBONUCLEASE T1
1I3F 2001-03-07 2.35 Ribonuclease T1 V89S mutant
1TRP 1994-04-30 2.4 X-RAY CRYSTALLOGRAPHIC AND CALORIMERIC STUDIES OF THE EFFECTS OF THE MUTATION TRP 59 TYR IN RIBONUCLEASE T1
3URP 2012-03-28 3.19 Re-refinement of PDB entry 5RNT - ribonuclease T1 with guanosine-3',5'-diphosphate and phosphate ion bound
5RNT 1993-01-15 3.2 X-RAY ANALYSIS OF CUBIC CRYSTALS OF THE COMPLEX FORMED BETWEEN RIBONUCLEASE T1 AND GUANOSINE-3',5'-BISPHOSPHATE

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR