Intact disulfide bonds decelerate the folding of ribonuclease T1.


Abstract

Disulfide bonds in a folding protein chain are equivalent to prematurely formed native-like tertiary interactions. We investigated whether the mechanism of protein folding is changed by the presence of disulfide bonds. As a model we used the S54G/P55N-variant of ribonuclease T1, a protein with two disulfide bonds and a single cis proline (Pro39), and we measured both the direct and the proline-limited folding reactions before and after breaking of the disulfide bonds. The folding kinetics were compared under refolding conditions, in the regions of the urea-induced unfolding transitions of the two forms, and under unfolding conditions. The kinetics in the transition regions were analyzed on the basis of a three-species mechanism and all microscopic rate constants of folding and of prolyl isomerization could be determined as a function of the urea concentration from the measured rates and amplitudes. These kinetic analyses indicated that the disulfide bonds can be rather unfavorable for the folding of S54G/P55N-ribonuclease T1. Under strongly native conditions they retard the rate-limiting trans-->cis isomerization of Pro39 because they allow the rapid formation of partially ordered structure prior to the proline-limited refolding reaction. Under unfolding conditions the isomerization of Pro39 is not affected. The direct unfolding and refolding reactions in the transition region of polypeptide chains with correct prolyl isomers are also decelerated when the disulfide bonds are present. These changes in the folding kinetics are possibly related to the decrease in chain flexibility that is caused by the disulfide bonds. A high flexibility is probably important throughout folding, and in the case of ribonuclease T1 a premature locking of tertiary contacts by intact disulfide bonds can interfere unfavorably with both the direct and the proline-limited folding reactions. Study holds ProTherm entries: 7478 Extra Details: protein folding; disulfide bonds; folding kinetics;,prolyl isomerization; protein stability

Submission Details

ID: qVox4yeF4

Submitter: Connie Wang

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

Version: 1

Publication Details
Mücke M;Schmid FX,J. Mol. Biol. (1994) Intact disulfide bonds decelerate the folding of ribonuclease T1. PMID:8014991
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
1IYY 2003-10-07 NMR STRUCTURE OF Gln25-RIBONUCLEASE T1, 24 STRUCTURES
1YGW 1997-10-08 NMR STRUCTURE OF RIBONUCLEASE T1, 34 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
4GSP 1998-08-12 1.65 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 7 DAYS
1I0X 2001-02-14 1.65 RIBONUCLEASE T1 IN COMPLEX WITH 2'GMP (FORM II CRYSTAL)
1RGA 1993-10-31 1.7 CRYSTAL STRUCTURE OF RNASE T1 WITH 3'-GMP AND GUANOSINE: A PRODUCT COMPLEX
4BIR 1998-07-15 1.7 RIBONUCLEASE T1: FREE HIS92GLN MUTANT
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
3BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
5BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
1RN4 1993-01-15 1.8 HIS92ALA MUTATION IN RIBONUCLEASE T1 INDUCES SEGMENTAL FLEXIBILITY. AN X-RAY STUDY
1DET 1996-07-11 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
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
3BIR 1997-12-31 1.8 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 BY ASN AND GLN SUBSTITUTIONS
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
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
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
1HZ1 2001-01-31 1.8 RIBONUCLEASE T1 V16A MUTANT IN COMPLEX WITH MG2+
3RNT 1989-10-15 1.8 CRYSTAL STRUCTURE OF GUANOSINE-FREE RIBONUCLEASE T1, COMPLEXED WITH VANADATE(V), SUGGESTS CONFORMATIONAL CHANGE UPON SUBSTRATE BINDING
2AAE 1994-01-31 1.8 THE ROLE OF HISTIDINE-40 IN RIBONUCLEASE T1 CATALYSIS: THREE-DIMENSIONAL STRUCTURES OF THE PARTIALLY ACTIVE HIS40LYS MUTANT
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
2HOH 1998-09-23 1.9 RIBONUCLEASE T1 (N9A MUTANT) COMPLEXED WITH 2'GMP
1BU4 1999-02-16 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
3GSP 1998-08-12 1.9 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS + 3'-GMP, 4 DAYS
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
7RNT 1993-01-15 1.9 CRYSTAL STRUCTURE OF THE TYR45TRP MUTANT OF RIBONUCLEASE T1 IN A COMPLEX WITH 2'-ADENYLIC ACID
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
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
1BVI 1998-09-23 1.9 RIBONUCLEASE T1 (WILDTYPE) COMPLEXED WITH 2'GMP
3HOH 1998-09-16 1.95 RIBONUCLEASE T1 (THR93GLN MUTANT) COMPLEXED WITH 2'GMP
1I2F 2001-03-07 1.95 Ribonuclease T1 V16A mutant, form II
1RHL 1998-10-14 1.95 RIBONUCLEASE T1 COMPLEXED WITH 2'GMP/G23A MUTANT
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
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.
2AAD 1994-01-31 2.0 THE ROLE OF HISTIDINE-40 IN RIBONUCLEASE T1 CATALYSIS: THREE-DIMENSIONAL STRUCTURES OF THE PARTIALLY ACTIVE HIS40LYS MUTANT
1RGL 1993-01-15 2.0 RNASE T1 MUTANT GLU46GLN BINDS THE INHIBITORS 2'GMP AND 2'AMP AT THE 3' SUBSITE
1FYS 2000-10-25 2.0 Ribonuclease T1 V16C mutant
7GSP 1998-03-18 2.0 RIBONUCLEASE T1/2',3'-CGPS, NON-PRODUCTIVE
5HOH 1998-09-23 2.0 RIBONUCLEASE T1 (ASN9ALA/THR93ALA DOUBLEMUTANT) COMPLEXED WITH 2'GMP
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
5BIR 1997-12-31 2.0 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 USING ASN AND GLN MUTATIONS
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
1GSP 1998-02-25 2.2 RIBONUCLEASE T1 COMPLEXED WITH 2',3'-CGPS, 1 DAY
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
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