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
ID: 32zkUmj23
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:42 p.m.
Version: 1
Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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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 |
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 |