Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1.


Abstract

The contribution of hydrogen bonding by peptide groups to the conformational stability of globular proteins was studied. One of the conserved residues in the microbial ribonuclease (RNase) family is an asparagine at position 39 in RNase Sa, 44 in RNase T1, and 58 in RNase Ba (barnase). The amide group of this asparagine is buried and forms two similar intramolecular hydrogen bonds with a neighboring peptide group to anchor a loop on the surface of all three proteins. Thus, it is a good model for the hydrogen bonding of peptide groups. When the conserved asparagine is replaced with alanine, the decrease in the stability of the mutant proteins is 2.2 (Sa), 1.8 (T1), and 2.7 (Ba) kcal/mol. When the conserved asparagine is replaced by aspartate, the stability of the mutant proteins decreases by 1.5 and 1.8 kcal/mol for RNases Sa and T1, respectively, but increases by 0.5 kcal/mol for RNase Ba. When the conserved asparagine was replaced by serine, the stability of the mutant proteins was decreased by 2.3 and 1.7 kcal/mol for RNases Sa and T1, respectively. The structure of the Asn 39 --> Ser mutant of RNase Sa was determined at 1.7 A resolution. There is a significant conformational change near the site of the mutation: (1) the side chain of Ser 39 is oriented differently than that of Asn 39 and forms hydrogen bonds with two conserved water molecules; (2) the peptide bond of Ser 42 changes conformation in the mutant so that the side chain forms three new intramolecular hydrogen bonds with the backbone to replace three hydrogen bonds to water molecules present in the wild-type structure; and (3) the loss of the anchoring hydrogen bonds makes the surface loop more flexible in the mutant than it is in wild-type RNase Sa. The results show that burial and hydrogen bonding of the conserved asparagine make a large contribution to microbial RNase stability and emphasize the importance of structural information in interpreting stability studies of mutant proteins. Study holds ProTherm entries: 3408, 3409, 3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 14209, 14210, 14211, 14212, 14213, 14214, 17860, 17861, 17862, 17863, 17864, 17865, 17866, 17867, 17868, 17869, 17870, 17871, 17872, 17873, 17874, 17875 Extra Details: delta(deltaG)=deltaTmdeltaSm,wt RNase Sa,and T1, Conformational stability, inter and intramolecular hydrogen bonds, conserved asparagine.

Submission Details

ID: rL6cmUY74

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:21 p.m.

Version: 1

Publication Details
Hebert EJ;Giletto A;Sevcik J;Urbanikova L;Wilson KS;Dauter Z;Pace CN,Biochemistry (1998) Contribution of a conserved asparagine to the conformational stability of ribonucleases Sa, Ba, and T1. PMID:9819211
Additional Information

Study Summary

Number of data points 101
Proteins Guanyl-specific ribonuclease Sa ; RIBONUCLEASE ; Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1
Unique complexes 8
Assays/Quantities/Protocols Experimental Assay: Tm prot_conc:~0.1 mg/mL, pH:2.0 ; Experimental Assay: dHvH prot_conc:~0.1 mg/mL, pH:2.0 ; Experimental Assay: ddG_H2O details:Additives ProTherm noted: average, units:kcal, pH:2.7, prot_conc:~0.1 mg/mL, buffer ; Experimental Assay: Cm buffers:glycine: 30 mM, prot_conc:~0.1 mg/mL, pH:2.7 ; Experimental Assay: m buffers:glycine: 30 mM, prot_conc:~0.1 mg/mL, pH:2.7 ; Experimental Assay: dG_H2O buffers:glycine: 30 mM, prot_conc:~0.1 mg/mL, pH:2.7 ; Experimental Assay: ddG_H2O details:Additives , buffers:MOPS: 30 mM, prot_conc:~0.1 mg/mL, units:kcal/mol, pH:7.0 ; Experimental Assay: Cm buffers:MOPS: 30 mM, prot_conc:~0.1 mg/mL, pH:7.0 ; Experimental Assay: m buffers:MOPS: 30 mM, prot_conc:~0.1 mg/mL, pH:7.0 ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, prot_conc:~0.1 mg/mL, pH:7.0 ; Experimental Assay: Tm prot_conc:~0.1 mg/mL, pH:7.0 ; Experimental Assay: dHvH prot_conc:~0.1 mg/mL, pH:7.0 ; Experimental Assay: ddG temp:50.8 C ; Experimental Assay: ddG temp:48.4 C ; Experimental Assay: ddG_H2O buffers:glycine: 30 mM, details:Additives , pH:2.7, units:kcal/mol ; Experimental Assay: Cm buffers:glycine: 30 mM, pH:2.7 ; Experimental Assay: m buffers:glycine: 30 mM, pH:2.7 ; Experimental Assay: dG_H2O buffers:glycine: 30 mM, pH:2.7 ; Experimental Assay: ddG_H2O pH:7.0, details:Additives , buffers:MOPS: 30 mM, units:kcal/mol ; Experimental Assay: Cm buffers:MOPS: 30 mM, pH:7.0 ; Experimental Assay: m buffers:MOPS: 30 mM, pH:7.0 ; Experimental Assay: dG_H2O buffers:MOPS: 30 mM, pH:7.0 ; Experimental Assay: Tm pH:7.0 ; Experimental Assay: dHvH pH:7.0 ; Derived Quantity: dTm prot_conc:~0.1 mg/mL, pH:2.0 ; Derived Quantity: dTm prot_conc:~0.1 mg/mL, pH:7.0 ; Derived Quantity: dTm pH:7.0
Libraries Mutations for sequence DVSGTVCLSALPPEATDTLNLIASDGPFPYSQDGVVFQNRESVLPTQSYGYYHEYTVITPGARTRGTRRIITGEATQEDYYTGDHYATFSLIDQTC ; Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT

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
1C54 2001-11-28 SOLUTION STRUCTURE OF RIBONUCLEASE SA
1IYY 2003-10-07 NMR STRUCTURE OF Gln25-RIBONUCLEASE T1, 24 STRUCTURES
1YGW 1997-10-08 NMR STRUCTURE OF RIBONUCLEASE T1, 34 STRUCTURES
1T2H 2004-12-21 1.0 Y81W mutant of RNase Sa from Streptomyces aureofaciens
1LNI 2002-07-31 1.0 CRYSTAL STRUCTURE ANALYSIS OF A RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS AT ATOMIC RESOLUTION (1.0 A)
4GHO 2013-08-14 1.1 Crystal Structure Analysis of Streptomyces aureofaciens Ribonuclease S24A mutant
1T2I 2004-12-21 1.1 T76W mutant of RNase Sa from Streptomyces aureofaciens
1ZGX 2006-08-08 1.13 Crystal structure of ribonuclease mutant
1RGE 1996-10-14 1.15 HYDROLASE, GUANYLORIBONUCLEASE
1RGH 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1RGF 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
1YNV 2005-07-19 1.2 Asp79 makes a large, unfavorable contribution to the stability of RNase Sa
1RGG 1996-10-14 1.2 HYDROLASE, GUANYLORIBONUCLEASE
4J5K 2014-05-28 1.23 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa Y51F mutant
1I0V 2001-02-14 1.23 Ribonuclease T1 in complex with 2'GMP (form I crystal)
1I8V 2001-09-19 1.25 CRYSTAL STRUCTURE OF RNASE SA Y80F MUTANT
4J5G 2014-05-28 1.31 Crystal structure analysis of Streptomyces aureofaciens ribonuclease Sa T95A mutant
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
1BOX 1999-12-29 1.6 N39S MUTANT OF RNASE SA FROM STREPTOMYCES AUREOFACIENS
3A5E 2010-08-04 1.6 Crystal structure of 5K RNase Sa
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)
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
1I70 2001-09-19 1.7 CRYSTAL STRUCTURE OF RNASE SA Y86F MUTANT
1GMP 1993-10-31 1.7 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
1Q9E 2004-03-23 1.7 RNase T1 variant with adenine specificity
1AY7 1999-03-02 1.7 RIBONUCLEASE SA COMPLEX WITH BARSTAR
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
5BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
1GMR 1993-10-31 1.77 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
3BU4 1998-09-23 1.77 RIBONUCLEASE T1 COMPLEX WITH 2'GMP
1SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
2SAR 1992-04-15 1.8 DETERMINATION AND RESTRAINED LEAST-SQUARES REFINEMENT OF THE CRYSTAL STRUCTURES OF RIBONUCLEASE SA AND ITS COMPLEX WITH 3'-GUANYLIC ACID AT 1.8 ANGSTROMS RESOLUTION
1UCI 2003-09-09 1.8 Mutants of RNase Sa
4BU4 1998-09-23 1.8 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
1FZU 2000-10-25 1.8 RNAse T1 V78A mutant
1HZ1 2001-01-31 1.8 RIBONUCLEASE T1 V16A MUTANT IN COMPLEX WITH MG2+
1DET 1996-07-11 1.8 RIBONUCLEASE T1 CARBOXYMETHYLATED AT GLU 58 IN COMPLEX WITH 2'GMP
6RNT 1993-01-15 1.8 CRYSTAL STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ADENOSINE 2'-MONOPHOSPHATE AT 1.8-ANGSTROMS RESOLUTION
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
8RNT 1993-01-15 1.8 STRUCTURE OF RIBONUCLEASE T1 COMPLEXED WITH ZINC(II) AT 1.8 ANGSTROMS RESOLUTION: A ZN2+.6H2O.CARBOXYLATE CLATHRATE
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
5GSP 1998-03-18 1.8 RIBONUCLEASE T1/3'-GMP, 9 WEEKS
3BIR 1997-12-31 1.8 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 BY ASN AND GLN SUBSTITUTIONS
1UCK 2003-09-09 1.8 Mutants of RNase Sa
1GMQ 1993-10-31 1.8 COMPLEX OF RIBONUCLEASE FROM STREPTOMYCES AUREOFACIENS WITH 2'-GMP AT 1.7 ANGSTROMS RESOLUTION
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
2GSP 1998-08-12 1.8 RIBONUCLEASE T1/2',3'-CGPS AND 3'-GMP, 2 DAYS
1UCJ 2003-09-09 1.81 Mutants of RNase Sa
1UCL 2003-09-09 1.82 Mutants of RNase Sa
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
7RNT 1993-01-15 1.9 CRYSTAL STRUCTURE OF THE TYR45TRP MUTANT OF RIBONUCLEASE T1 IN A COMPLEX WITH 2'-ADENYLIC ACID
1BU4 1999-02-16 1.9 RIBONUCLEASE 1 COMPLEX WITH 2'GMP
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
1LRA 1994-01-31 1.9 CRYSTALLOGRAPHIC STUDY OF GLU 58 ALA RNASE T1(ASTERISK)2'-GUANOSINE MONOPHOSPHATE AT 1.9 ANGSTROMS RESOLUTION
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
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
2HOH 1998-09-23 1.9 RIBONUCLEASE T1 (N9A MUTANT) COMPLEXED 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
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
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
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
7GSP 1998-03-18 2.0 RIBONUCLEASE T1/2',3'-CGPS, NON-PRODUCTIVE
1FYS 2000-10-25 2.0 Ribonuclease T1 V16C mutant
1RSN 1995-12-07 2.0 RIBONUCLEASE (RNASE SA) (E.C.3.1.4.8) COMPLEXED WITH EXO-2',3'-CYCLOPHOSPHOROTHIOATE
5HOH 1998-09-23 2.0 RIBONUCLEASE T1 (ASN9ALA/THR93ALA DOUBLEMUTANT) COMPLEXED 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.
5BIR 1997-12-31 2.0 DISECTING HISTIDINE INTERACTIONS IN RIBONUCLEASE T1 USING ASN AND GLN MUTATIONS
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
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
6GSP 1998-03-18 2.2 RIBONUCLEASE T1/3'-GMP, 15 WEEKS
4RNT 1992-01-15 2.2 HIS 92 ALA MUTATION IN RIBONUCLEASE T1 INDUCES SEGMENTAL FLEXIBILITY. AN X-RAY STUDY
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
1TRQ 1994-04-30 2.3 X-RAY CRYSTALLOGRAPHIC AND CALORIMERIC STUDIES OF THE EFFECTS OF THE MUTATION TRP 59 TYR IN RIBONUCLEASE T1
2BIR 1997-06-16 2.3 ADDITIVITY OF SUBSTRATE BINDING IN RIBONUCLEASE T1 (Y42A MUTANT)
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 Sa P05798 RNSA_KITAU
100.0 Guanyl-specific ribonuclease T1 P00651 RNT1_ASPOR