Decoupling of melting domains in immobilized ribonuclease A.

Abstract

Ribonuclease A has been immobilized on silica beads through glutaraldeyde-mediated chemical coupling in order to improve the stability of the protein against thermal denaturation. The thermodynamic and binding properties of the immobilized enzyme have been studied and compared with those of the free enzyme. The parameters describing the binding of the inhibitor 3'-CMP (Ka and delta H) as monitored by spectrophotometry and calorimetry were not significantly affected after immobilization. Conversely both the stability and unfolding mechanism drastically changed. Thermodynamic analysis of the DSC data suggests that uncoupling of protein domains has occurred as a consequence of the immobilization. The two state approximation of the protein unfolding process is not longer valid for the immobilized RNase. Protein stability strongly depends on the hydrophobicity properties of the support surface as well as on the presence of the inhibitor and pH. For example, after immobilization on a highly hydrophobic surface, the enzyme is partially in the unfolded state. The binding of a ligand is able to reorganize the protein structure into a native-like conformation. The refolding rates are different for the two protein domains and vary as a function of pH and presence of the inhibitor 3'-CMP. Study holds ProTherm entries: 11304, 11305 Extra Details: enzymes; protein; immobilization; microcalorimetry;,protien melting domains; protein DSC

Submission Details

ID: 32zkUmj23

Submitter: Connie Wang

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

Version: 1

Publication Details
Rialdi G;Battistel E,Proteins (1994) Decoupling of melting domains in immobilized ribonuclease A. PMID:8090706
Additional Information

Study Summary

Number of data points 4
Proteins Ribonuclease pancreatic ; Ribonuclease pancreatic
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: dHcal buffers:bis-Tris-HCl: 50 mM, pH:7.0 ; Experimental Assay: Tm buffers:bis-Tris-HCl: 50 mM, pH:7.0 ; Experimental Assay: dHcal pH:5.0, buffers:acetate: 50 mM ; Experimental Assay: Tm pH:5.0, buffers:acetate: 50 mM
Libraries 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
6QMN 2019-02-07T00:00:00+0000 2.31 Crystal structure of a Ribonuclease A-Onconase chimera
3OQY 2010-09-04T00:00:00+0000 1.49 Semi-synthetic ribonuclease S: para-cyano-phenylalanine at position 8
3OQY 2010-09-04T00:00:00+0000 1.49 Semi-synthetic ribonuclease S: para-cyano-phenylalanine at position 8
1KF4 2001-11-19T00:00:00+0000 1.1 Atomic Resolution Structure of RNase A at pH 6.3
3DH6 2008-06-17T00:00:00+0000 1.6 Crystal structure of bovine pancreatic ribonuclease A variant (V47A)
1EOS 2000-03-24T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF RIBONUCLEASE A COMPLEXED WITH URIDYLYL(2',5')GUANOSINE (PRODUCTIVE BINDING)
3RN3 1991-10-30T00:00:00+0000 1.45 SEGMENTED ANISOTROPIC REFINEMENT OF BOVINE RIBONUCLEASE A BY THE APPLICATION OF THE RIGID-BODY TLS MODEL
4G8Y 2012-07-23T00:00:00+0000 1.8 Crystal structure of Ribonuclease A in complex with 5b
5JLG 2016-04-27T00:00:00+0000 1.79 The X-ray structure of the adduct formed in the reaction between bovine pancreatic ribonuclease and compound I, a piano-stool organometallic Ru(II) arene compound containing an O,S-chelating ligand
4G8V 2012-07-23T00:00:00+0000 1.7 Crystal structure of Ribonuclease A in complex with 5a

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