Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site.


Abstract

To clarify the structural role of Phe46 inside the hydrophobic core of bovine pancreatic ribonuclease A (RNase A), thermal and pressure unfolding of wild-type RNase A and three mutant forms (F46V, F46E, and F46K) were analyzed by fourth-derivative UV absorbance spectroscopy. All the mutants, as well as the wild type, exhibited a two-state transition during both thermal and pressure unfolding, and both T(m) and P(m) decreased markedly when Phe46 was replaced with valine, glutamic acid, or lysine. The strongest effect was on the F46K mutant and the weakest on F46V. Both unfolding processes produced identical blue shifts in the fourth-derivative spectra, indicating that the tyrosine residues are similarly exposed in the temperature- and pressure-induced unfolded states. A comparison of Gibbs free energies determined from the pressure and temperature unfoldings, however, gave DeltaG(p)/DeltaG(t) ratios (r) of 1.7 for the wild type and 0.92 +/- 0.03 for the mutants. Furthermore, the DeltaV value for each mutant was larger than that for the wild type. CD spectra and activity measurements showed no obvious major structural differences in the folded state, indicating that the structures of the Phe46 mutants and wild type differ in the unfolded state. We propose a model in which Phe46 stabilizes the hydrophobic core at the boundary between two structural domains. Mutation of Phe46 decreases protein stability by weakening the unfolding cooperativity between these domains. This essential function of Phe46 in RNase A stability indicates that it belongs to a chain-folding initiation site. Study holds ProTherm entries: 14917, 14918, 14919, 14920, 14921, 14922, 14923, 14924 Extra Details: hydrophobic core; two-state transition; pressure unfolding; structural domains

Submission Details

ID: f546P2zr

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:45 p.m.

Version: 1

Publication Details
Chatani E;Nonomura K;Hayashi R;Balny C;Lange R,Biochemistry (2002) Comparison of heat- and pressure-induced unfolding of ribonuclease a: the critical role of Phe46 which appears to belong to a new hydrophobic chain-folding initiation site. PMID:11926818
Additional Information

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
1A2W 1998-01-12T00:00:00+0000 2.1 CRYSTAL STRUCTURE OF A 3D DOMAIN-SWAPPED DIMER OF BOVINE PANCREATIC RIBONUCLEASE A
1A5P 1998-02-17T00:00:00+0000 1.6 C[40,95]A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1A5Q 1998-02-17T00:00:00+0000 2.3 P93A VARIANT OF BOVINE PANCREATIC RIBONUCLEASE A
1AFK 1997-03-08T00:00:00+0000 1.7 CRYSTAL STRUCTURE OF RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE-3'-PHOSPHATE
1AFL 1997-03-08T00:00:00+0000 1.7 RIBONUCLEASE A IN COMPLEX WITH 5'-DIPHOSPHOADENOSINE 2'-PHOSPHATE AT 1.7 ANGSTROM RESOLUTION
1AFU 1997-03-14T00:00:00+0000 2.0 STRUCTURE OF RIBONUCLEASE A AT 2.0 ANGSTROMS FROM MONOCLINIC CRYSTALS
1AQP 1997-07-31T00:00:00+0000 2.0 RIBONUCLEASE A COPPER COMPLEX
1B6V 1999-01-18T00:00:00+0000 2.0 CRYSTAL STRUCTURE OF A HYBRID BETWEEN RIBONUCLEASE A AND BOVINE SEMINAL RIBONUCLEASE
1BEL 1995-12-21T00:00:00+0000 1.6 HYDROLASE PHOSPHORIC DIESTER, RNA

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