Cysteine-free Rop: a four-helix bundle core mutant has wild-type stability and structure but dramatically different unfolding kinetics.


Abstract

Cysteine residues can complicate the folding and storage of proteins due to improper formation of disulfide bonds or oxidation of residues that are natively reduced. Wild-type Rop is a homodimeric four-helix bundle protein and an important model for protein design in the understanding of protein stability, structure and folding kinetics. In the native state, Rop has two buried, reduced cysteine residues in its core, but these are prone to oxidation in destabilized variants, particularly upon extended storage. To circumvent this problem, we designed and characterized a Cys-free variant of Rop, including solving the 2.3 A X-ray crystal structure. We show that the C38A C52V variant has similar structure, stability and in vivo activity to wild-type Rop, but that it has dramatically faster unfolding kinetics like virtually every other mutant of Rop that has been characterized. This cysteine-free Rop has already proven useful for studies on solution topology and on the relationship of core mutations to stability. It also suggests a general strategy for removal of pairs of Cys residues in proteins, both to make them more experimentally tractable and to improve their storage properties for therapeutic or industrial purposes. Study holds ProTherm entries: 25701, 25702, 25703, 25704, 25705, 25706, 25707 Extra Details: Rop; four-helix bundle; cysteine-free; unfolding kinetics

Submission Details

ID: avqWL8w93

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:56 p.m.

Version: 1

Publication Details
Hari SB;Byeon C;Lavinder JJ;Magliery TJ,Protein Sci. (2010) Cysteine-free Rop: a four-helix bundle core mutant has wild-type stability and structure but dramatically different unfolding kinetics. PMID:20095056
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
1RPR 1994-01-31 THE STRUCTURE OF COLE1 ROP IN SOLUTION
1NKD 1999-03-23 1.09 ATOMIC RESOLUTION (1.07 ANGSTROMS) STRUCTURE OF THE ROP MUTANT <2AA>
4DO2 2013-02-13 1.4 Crystal Structure of the Rop protein mutant D30P/A31G at resolution 1.4 resolution.
1RPO 1995-02-14 1.4 RESTORED HEPTAD PATTERN CONTINUITY DOES NOT ALTER THE FOLDING OF A 4-ALPHA-HELICAL BUNDLE
2IJK 2007-10-16 1.55 Structure of a Rom protein dimer at 1.55 angstrom resolution
1ROP 1992-07-15 1.7 STRUCTURE OF THE COL*E1 ROP PROTEIN AT 1.7 ANGSTROMS RESOLUTION
2IJH 2007-10-16 1.8 Crystal structure analysis of ColE1 ROM mutant F14W
1B6Q 1999-07-09 1.8 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN
1GTO 1997-01-27 1.82 HIGH RESOLUTION STRUCTURE OF A HYPERSTABLE HELICAL BUNDLE PROTEIN MUTANT
2IJJ 2007-10-16 1.9 Crystal structure analysis of ColE1 ROM mutant F14Y
1GMG 2002-09-12 1.9 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN, MONOCLINIC FORM
1F4N 2001-01-10 1.9 C2 CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
3K79 2010-02-02 1.96 C38A, C52V Cysteine-Free Variant of Rop (Rom)
1QX8 2004-09-28 2.02 Crystal structure of a five-residue deletion mutant of the Rop protein
1F4M 2001-01-10 2.25 P3(2) CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
2IJI 2007-10-16 2.3 Structure of F14H mutant of ColE1 Rom protein
2GHY 2006-05-30 2.5 Novel Crystal Form of the ColE1 Rom Protein
1YO7 2005-02-15 2.8 Re-engineering topology of the homodimeric ROP protein into a single-chain 4-helix bundle

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Regulatory protein rop P03051 ROP_ECOLX