Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori.


Abstract

Helicobacter pylori cysteine-rich proteins (Hcps) are disulfide-containing repeat proteins. The repeating unit is a 36-residue, disulfide-bridged, helix-loop-helix motif. We use the protein HcpB, which has four repeats and four disulfide bridges arrayed in tandem, as a model to determine the thermodynamic stability of a disulfide-rich repeat protein and to study the formation and the contribution to stability of the disulfide bonds. When the disulfide bonds are intact, the chemical unfolding of HcpB at pH 5 is cooperative and can be described by a two-state reaction. Thermal unfolding is reversible between pH 2 and 5 and irreversible at higher pH 5. Differential scanning calorimetry shows noncooperative structural changes preceding the main thermal unfolding transition. Unfolding of the oxidized protein is not an all-or-none two-state process, and the disulfide bonds prevent complete unfolding of the polypeptide chain. The reduced protein is significantly less stable and does not unfold in a cooperative way. During oxidative refolding of the fully reduced protein, all the possible disulfide intermediates with a correct disulfide bond are formed. Formation of "wrong" (non-native) disulfide bonds could not be demonstrated, indicating that the reduced protein already has some partial repeating structure. There is a major folding intermediate with disulfides in the second, third, and fourth repeat and reduced cysteines in the first repeat. Disulfide formation in the first repeat limits the overall rate of oxidative refolding and contributes about half of the thermodynamic stability to native HcpB, estimated as 27 kJ mol(-1) at 25 degrees C and pH 7. The high contribution to stability of the first repeat may be explained by the repeat acting as a cap to protect the hydrophobic interior of the molecule. Study holds ProTherm entries: 19777, 19778, 19779, 19780, 19781, 19782, 19783, 19784, 19785, 19786, 19787 Extra Details: oxidized form; 15 mM DTT was added in the experiment. cysteine-rich proteins; helix-loop-helix motif; disulfide bonds; noncooperative structural changes

Submission Details

ID: Cfdh7CJG4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:52 p.m.

Version: 1

Publication Details
Devi VS;Sprecher CB;Hunziker P;Mittl PR;Bosshard HR;Jelesarov I,Biochemistry (2006) Disulfide formation and stability of a cysteine-rich repeat protein from Helicobacter pylori. PMID:16460007
Additional Information

Study Summary

Number of data points 26
Proteins Beta-lactamase HcpB ; Beta-lactamase HcpB
Unique complexes 1
Assays/Quantities/Protocols Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: dHcal pH:5.0 ; Experimental Assay: Tm prot_conc:150 microM, pH:5.0, details:Additives ; Experimental Assay: dHvH prot_conc:150 microM, pH:5.0, details:Additives ; Experimental Assay: dHcal pH:4.0 ; Experimental Assay: Tm prot_conc:150 microM, pH:4.0, details:Additives ; Experimental Assay: dHvH prot_conc:150 microM, pH:4.0, details:Additives ; Experimental Assay: dHcal pH:3.0 ; Experimental Assay: Tm prot_conc:150 microM, pH:3.0, details:Additives ; Experimental Assay: dHvH prot_conc:150 microM, pH:3.0, details:Additives ; Experimental Assay: dHcal pH:2.0 ; Experimental Assay: Tm prot_conc:150 microM, pH:2.0, details:Additives ; Experimental Assay: dHvH prot_conc:150 microM, details:Additives , pH:2.0 ; Experimental Assay: Tm pH:5.0, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:5.0, details:Additives DTT (15 mM), ; Experimental Assay: Tm pH:4.0, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:4.0, details:Additives DTT (15 mM), ; Experimental Assay: Tm pH:3.5, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:3.5, details:Additives DTT (15 mM), ; Experimental Assay: Tm pH:3.0, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:3.0, details:Additives DTT (15 mM), ; Experimental Assay: Tm pH:2.4, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:2.4, details:Additives DTT (15 mM), ; Experimental Assay: Tm pH:2.0, details:Additives DTT (15 mM), ; Experimental Assay: dHvH pH:2.0, details:Additives DTT (15 mM),
Libraries Mutations for sequence MVGGGTVKKDLKKAIQYYVKACELNEMFGCLSLVSNSQINKQKLFQYLSKACELNSGNGCRFLGDFYENGKYVKKDLRKAAQYYSKACGLNDQDGCLILGYKQYAGKGVVKNEKQAVKTFEKACRLGSEDACGILNNY

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
1KLX 2002-09-11 1.95 Helicobacter pylori cysteine rich protein B (hcpB)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
100.0 Beta-lactamase HcpB O25103 HCPB_HELPY