Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A.


Abstract

In order to probe changes in the structural stability induced by the introduction of hydrophobic groups into proteins, the amino groups of ribonuclease A and chymotrypsinogen A were reductively alkylated by reaction with various aliphatic aldehydes, formaldehyde, acetaldehyde, n-butylaldehyde and n-hexylaldehyde, and their thermal stabilities were investigated by differential scanning calorimetry (DSC) at different acidic pH values. Ribonuclease A was thermally unstabilized by reductive alkylation, while chymotrypsinogen A was slightly stabilized, depending on both the size of the introduced alkyl groups and the extent of modification. These observations suggest that the effects induced by alkylation involve not only steric hindrance due to the entering bulky groups but also certain other factors such as the participation of the chemically introduced alkyl groups in hydrophobic interactions. Study holds ProTherm entries: 11525, 11526, 11527, 11528, 11529, 11530, 11531, 11532, 11533, 11534 Extra Details: alkylation; chemical modification; denaturation; DSC; protein; stability

Submission Details

ID: MTE6YsTW4

Submitter: Connie Wang

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

Version: 1

Publication Details
Fujita Y;Noda Y,Int. J. Pept. Protein Res. (1991) Effect of reductive alkylation on thermal stability of ribonuclease A and chymotrypsinogen A. PMID:1802862
Additional Information

Study Summary

Number of data points 20
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic ; Chymotrypsinogen A ; Calnexin
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: dCp buffers:glycine: 0.01 M ; Experimental Assay: dG buffers:glycine: 0.01 M ; Experimental Assay: dCp buffers:glycine: 0.05 M ; Experimental Assay: dG buffers:glycine: 0.05 M ; Experimental Assay: dHcal pH:3.5, buffers:glycine: 0.01 M ; Experimental Assay: Tm pH:3.5, buffers:glycine: 0.01 M ; Experimental Assay: dHcal pH:3.0, buffers:glycine: 0.01 M ; Experimental Assay: Tm pH:3.0, buffers:glycine: 0.01 M ; Experimental Assay: dHcal pH:2.5, buffers:glycine: 0.01 M ; Experimental Assay: Tm pH:2.5, buffers:glycine: 0.01 M ; Experimental Assay: dHcal pH:2.0, buffers:glycine: 0.01 M ; Experimental Assay: Tm pH:2.0, buffers:glycine: 0.01 M ; Experimental Assay: dHcal pH:4.0, buffers:glycine: 0.05 M ; Experimental Assay: Tm pH:4.0, buffers:glycine: 0.05 M ; Experimental Assay: dHcal buffers:glycine: 0.05 M, pH:3.0 ; Experimental Assay: Tm buffers:glycine: 0.05 M, pH:3.0 ; Experimental Assay: dHcal pH:2.5, buffers:glycine: 0.05 M ; Experimental Assay: Tm pH:2.5, buffers:glycine: 0.05 M ; Experimental Assay: dHcal buffers:glycine: 0.05 M, pH:2.0 ; Experimental Assay: Tm buffers:glycine: 0.05 M, pH:2.0
Libraries Mutations for sequence SKSKPDTSAPTSPKVTYKAPVPSGEVYFADSFDRGTLSGWILSKAKKDDTDDEIAKYDGKWEVDEMKETKLPGDKGLVLMSRAKHHAISAKLNKPFLFDTKPLIVQYEVNFQNGIECGGAYVKLLSKTPELNLDQFHDKTPYTIMFGPDKCGEDYKLHFIFRHKNPKTGVYEEKHAKRPDADLKTYFTDKKTHLYTLILNPDNSFEILVDQSIVNSGNLLNDMTPPVNPSREIEDPEDQKPEDWDERPKIPDPDAVKPDDWNEDAPAKIPDEEATKPDGWLDDEPEYVPDPDAEKPEDWDEDMDGEWEAPQIANPKCESAPGCGVWQRPMIDNPNYKGKWKPPMIDNPNYQGIWKPRKIPNPDFFEDLEPFKMTPFSAIGLELWSMTSDIFFDNFIVCGDRRVVDDWANDGWGLKKAADGAAEP ; Mutations for sequence KETAAAKFERQHMDSSTSAASSSNYCNQMMKSRNLTKDRCKPVNTFVHESLADVQAVCSQKNVACKNGQTNCYQSYSTMSITDCRETGSSKYPNCAYKTTQANKHIIVACEGNPYVPVHFDASV

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
1JHN 2001-06-28T00:00:00+0000 2.9 Crystal Structure of the Lumenal Domain of Calnexin
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
1AB9 1997-02-05T00:00:00+0000 1.6 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN
1AB9 1997-02-05T00:00:00+0000 1.6 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN
1AB9 1997-02-05T00:00:00+0000 1.6 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN
1ACB 1991-11-08T00:00:00+0000 2.0 CRYSTAL AND MOLECULAR STRUCTURE OF THE BOVINE ALPHA-CHYMOTRYPSIN-EGLIN C COMPLEX AT 2.0 ANGSTROMS RESOLUTION
1AFQ 1997-03-12T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN COMPLEXED WITH A SYNTHETIC INHIBITOR
1AFQ 1997-03-12T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN COMPLEXED WITH A SYNTHETIC INHIBITOR
1AFQ 1997-03-12T00:00:00+0000 1.8 CRYSTAL STRUCTURE OF BOVINE GAMMA-CHYMOTRYPSIN COMPLEXED WITH A SYNTHETIC INHIBITOR
1CA0 1997-01-23T00:00:00+0000 2.1 BOVINE CHYMOTRYPSIN COMPLEXED TO APPI

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.0 Ribonuclease pancreatic Q29606 RNAS1_ORYLE
90.3 Ribonuclease pancreatic P00662 RNAS1_GIRCA
92.7 Ribonuclease pancreatic P00668 RNAS1_ANTAM
95.2 Ribonuclease pancreatic P00660 RNAS1_CONTA
93.5 Ribonuclease pancreatic P07848 RNAS1_EUDTH
96.0 Ribonuclease pancreatic P07847 RNAS1_AEPME
95.2 Ribonuclease pancreatic P00657 RNAS1_BUBBU
96.8 Ribonuclease pancreatic P67927 RNAS1_SHEEP
96.8 Ribonuclease pancreatic P67926 RNAS1_CAPHI
100.0 Ribonuclease pancreatic P61823 RNAS1_BOVIN
100.0 Ribonuclease pancreatic P61824 RNAS1_BISBI
100.0 Chymotrypsinogen A P00766 CTRA_BOVIN
96.4 Calnexin P35565 CALX_RAT
97.6 Calnexin P35564 CALX_MOUSE
96.1 Calnexin P27824 CALX_HUMAN
96.4 Calnexin Q5R440 CALX_PONAB
99.8 Calnexin P24643 CALX_CANLF