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

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
Sequence Assay Result Units