Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine.


Abstract

The refolding of ribonuclease T1 involves two major slow processes that exhibit properties of prolyl isomerization reactions. A comparison of the wild-type protein and a designed variant where the cis Ser54-Pro55 bond was replaced by a Gly54-Asn55 bond indicated that the faster of these reactions is the isomerization of Pro55. Here we report the replacement of the other cis proline of ribonuclease T1 at position 39 by alanine. The Pro39Ala variant is similar to the wild-type protein in secondary and tertiary structure, and the enzymatic activity towards RNA and a dinucleotide substrate remains almost unchanged. The fluorescence emission of the single Trp59 is lowered by the Pro39Ala substitution, probably because Trp59 is in close contact to Pro39 in wild-type ribonuclease T1. Unlike the substitution of cis Pro55, the Pro39Ala mutation is strongly destabilizing and reduces the Gibbs free energy of the folded protein by about 20 kJ/mol. Pro39 is buried in native RNase T1 and located near the active site. The observed destabilization could originate from the presence of a cis alanyl bond in the Pro39Ala variant or from a local distortion caused by the incorporation of a trans alanyl peptide bond in the interior of the protein. In the refolding kinetics the replacement of Pro39 leads to a disappearance of the fast-refolding species. Refolding still involves two consecutive slow steps. The first and faster step could be the isomerization of the remaining cis Pro55. The second, very slow step is a novel reaction that appears to have no counterpart in the refolding of the wild-type protein. All mutant molecules must undergo this reaction before reaching the native state. These major changes in the folding kinetics strongly indicate that cis-Pro39 is indeed of major importance for the folding of the wild-type protein. They indicate, moreover, that some new feature of protein folding kinetics is observed in these studies of the Pro39Ala variant. Study holds ProTherm entries: 2381, 2382, 2383, 2384, 2385, 2386, 2387 Extra Details: protein folding; folding kinetics; proline substitution;,prolyl cis/trans-isomerization

Submission Details

ID: vF92Vtss3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:18 p.m.

Version: 1

Publication Details
Mayr LM;Landt O;Hahn U;Schmid FX,J. Mol. Biol. (1993) Stability and folding kinetics of ribonuclease T1 are strongly altered by the replacement of cis-proline 39 with alanine. PMID:8515459
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
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
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+
1I3I 2001-03-07 1.76 Ribonuclease T1 V78T mutant
5BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
3BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
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
1BIR 1996-08-17 1.8 RIBONUCLEASE T1, PHE 100 TO ALA MUTANT COMPLEXED WITH 2' GMP
3RNT 1989-10-15 1.8 CRYSTAL STRUCTURE OF GUANOSINE-FREE RIBONUCLEASE T1, COMPLEXED WITH VANADATE(V), SUGGESTS CONFORMATIONAL CHANGE UPON SUBSTRATE BINDING
1I2E 2001-03-07 1.8 Ribonuclease T1 V16A mutant, form I
2GSP 1998-08-12 1.8 RIBONUCLEASE T1/2',3'-CGPS AND 3'-GMP, 2 DAYS
1DET 1996-07-11 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
3BIR 1997-12-31 1.8 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 BY ASN AND GLN SUBSTITUTIONS
6RNT 1993-01-15 1.8 CRYSTAL STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ADENOSINE 2'-MONOPHOSPHATE AT 1.8-ANGSTROMS RESOLUTION
8RNT 1993-01-15 1.8 STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ZINC(II) AT 1.8 ANGSTROMS RESOLUTION: A ZN2+.6H2O.CARBOXYLATE CLATHRATE
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
1HZ1 2001-01-31 1.8 RIBONUCLEASE T1 V16A MUTANT IN COMPLEX WITH MG2+
1RN4 1993-01-15 1.8 HIS92ALA MUTATION IN RIBONUCLEASE T1 INDUCES SEGMENTAL FLEXIBILITY. AN X-RAY STUDY
1FZU 2000-10-25 1.8 RNAse T1 V78A mutant
4BU4 1998-09-23 1.8 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
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
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
3GSP 1998-08-12 1.9 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 4 DAYS
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
1LOW 2002-08-21 1.9 X-ray structure of the H40A mutant of Ribonuclease T1 complexed with 3'-guanosine monophosphate
1LRA 1994-01-31 1.9 CRYSTALLOGRAPHIC STUDY OF GLU 58 ALA RNASE T1(ASTERISK)2'-GUANOSINE MONOPHOSPHATE AT 1.9 ANGSTROMS RESOLUTION
2HOH 1998-09-23 1.9 RIBONUCLEASE T1 (N9A MUTANT) COMPLEXED WITH 2'GMP
1BVI 1998-09-23 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
1BU4 1999-02-16 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
7RNT 1993-01-15 1.9 CRYSTAL STRUCTURE OF THE TYR45TRP MUTANT OF RIBONUCLEASE T1 IN A COMPLEX WITH 2'-ADENYLIC ACID
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
1RHL 1998-10-14 1.95 RIBONUCLEASE T1 COMPLEXED WITH 2'GMP/G23A MUTANT
3SYU 2012-03-28 1.95 Re-refined coordinates for pdb entry 1det - ribonuclease T1 carboxymethylated at GLU 58 in complex with 2'GMP
1I2F 2001-03-07 1.95 Ribonuclease T1 V16A mutant, form II
5HOH 1998-09-23 2.0 RIBONUCLEASE T1 (ASN9ALA/THR93ALA DOUBLEMUTANT) COMPLEXED WITH 2'GMP
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
2AAD 1994-01-31 2.0 THE ROLE OF HISTIDINE-40 IN RIBONUCLEASE T1 CATALYSIS: THREE-DIMENSIONAL STRUCTURES OF THE PARTIALLY ACTIVE HIS40LYS MUTANT
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
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.
1RGL 1993-01-15 2.0 RNASE T1 MUTANT GLU46GLN BINDS THE INHIBITORS 2'GMP AND 2'AMP AT THE 3' SUBSITE
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