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
1B6Q 1999-01-16T00:00:00+0000 1.8 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN
1F4M 2000-06-08T00:00:00+0000 2.25 P3(2) CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
1F4N 2000-06-08T00:00:00+0000 1.9 C2 CRYSTAL STRUCTURE OF ALA2ILE2-6, A VERSION OF ROP WITH A REPACKED HYDROPHOBIC CORE AND A NEW FOLD.
1GMG 2001-09-13T00:00:00+0000 1.9 ALANINE 31 PROLINE MUTANT OF ROP PROTEIN, MONOCLINIC FORM
1GTO 1996-04-23T00:00:00+0000 1.82 HIGH RESOLUTION STRUCTURE OF A HYPERSTABLE HELICAL BUNDLE PROTEIN MUTANT
1NKD 1997-09-23T00:00:00+0000 1.09 ATOMIC RESOLUTION (1.07 ANGSTROMS) STRUCTURE OF THE ROP MUTANT <2AA>
1QX8 2003-09-04T00:00:00+0000 2.02 Crystal structure of a five-residue deletion mutant of the Rop protein
1ROP 1991-04-02T00:00:00+0000 1.7 STRUCTURE OF THE COL*E1 ROP PROTEIN AT 1.7 ANGSTROMS RESOLUTION
1RPO 1994-08-25T00:00:00+0000 1.4 RESTORED HEPTAD PATTERN CONTINUITY DOES NOT ALTER THE FOLDING OF A 4-ALPHA-HELICAL BUNDLE
1RPR 1991-10-09T00:00:00+0000 0 THE STRUCTURE OF COLE1 ROP IN SOLUTION

Relevant UniProtKB Entries

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