Hydrophobic core manipulations in ribonuclease T1.


Abstract

Differential scanning calorimetry, urea denaturation, and X-ray crystallography were combined to study the structural and energetic consequences of refilling an engineered cavity in the hydrophobic core of RNase T1 with CH(3), SH, and OH groups. Three valines that cluster together in the major hydrophobic core of T1 were each replaced with Ala, Ser, Thr, and Cys. Compared to the wild-type protein, all these mutants reduce the thermodynamic stability of the enzyme considerably. The relative order of stability at all three positions is as follows: Val > Ala approximately equal to Thr > Ser. The effect of introducing a sulfhydryl group is more variable. Surprisingly, a Val --> Cys mutation in a hydrophobic environment can be as or even more destabilizing than a Val --> Ser mutation. Furthermore, our results reveal that the penalty for introducing an OH group into a hydrophobic cavity is roughly the same as the gain obtained from filling the cavity with a CH(3) group. The inverse equivalence of the behavior of hydroxyl and methyl groups seems to be crucial for the unique three-dimensional structure of the proteins. The importance of negative design elements in this context is highlighted. Study holds ProTherm entries: 11637, 11638, 11639, 11640, 11641, 11642, 11643, 11644, 11645, 11646, 11647, 11648, 11649, 11650, 11651, 11652, 11653, 11654, 11655, 11656, 11657, 11658, 11659, 11660, 11661, 11662, 11663, 11664, 11665, 11666, 11667, 11668, 11669 Extra Details: cavity; hydrophobic core; sulfhydryl group; hydroxyl; methyl groups

Submission Details

ID: oTb25wZJ4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:42 p.m.

Version: 1

Publication Details
De Vos S;Backmann J;Prévost M;Steyaert J;Loris R,Biochemistry (2001) Hydrophobic core manipulations in ribonuclease T1. PMID:11513591
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