Equilibrium denaturation of human growth hormone and its cysteine-modified forms.


Abstract

The equilibrium denaturation of human growth hormone (hGH) derived from heterologous gene expression in Escherichia coli was studied. Denaturation was measured by ultraviolet absorbance, intrinsic fluorescence, far ultraviolet circular dichroism, and size exclusion chromatography. The denaturation transitions obtained from each method of detection were coincident, indicating a two-state denaturation mechanism. The denaturation transitions were independent of the concentration of protein. The Gibbs free energy of unfolding is 14.5 +/- 1 kcal/mol. Human growth hormone contains two disulfide bridges between residues 53-165 (large loop) and 182-189 (small loop). The small loop was selectively reduced and cysteines alkylated with iodoacetic acid or iodoacetamide. The tetra-S-carbamidomethylated and tetra-S-carboxymethylated derivatives were also prepared. All S-alkylated hGH forms were indistinguishable from the native conformations in the absence of denaturant by far ultraviolet circular dichroism. The circular dichroism-detected equilibrium denaturation of each derivative was determined and the Gibbs free energy of unfolding of the tetra-S-modified forms was 5.3 +/- 0.5 kcal/mol and of the di-S-alkylated derivatives was 11.2 +/- 0.8 kcal/mol. These results for hGH are different than previously obtained results for bovine, ovine, and rat growth hormones. Stable equilibrium intermediates have been identified for these non-human species of growth hormone. The stable intermediates observed in the denaturation of reduced, alkylated hGH or nonhunam growth hormones are similar and characterized as compact, helical, lacking native-like tertiary structure, and having a tendency to aggregate. The apparent absence of intermediates in the folding of oxidized hGH is due to the relative instability of intermediates compared with their native structures. The hGH conformation is at least 5 kcal/mol more stable than the growth hormones from other species. Reduction and alkylation of the disulfide bridges of hGH diminish the stability differences between the native and intermediate states, such that the denaturation behavior is similar to the nonhuman growth hormones with well-populated intermediates. Most proteins do not demonstrate equilibrium folding intermediates presumably because intermediates are only marginally stable in conditions that disrupt the native state. The folding results with hGH and alkylated hGH substantiate this. Study holds ProTherm entries: 5172 Extra Details: two-state denaturation mechanism; disulfide bridges;,stable intermediates; native-like tertiary structure

Submission Details

ID: MKDubnaD

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:28 p.m.

Version: 1

Publication Details
Brems DN;Brown PL;Becker GW,J. Biol. Chem. (1990) Equilibrium denaturation of human growth hormone and its cysteine-modified forms. PMID:2180927
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
5OHD 2018-04-11 Putative inactive (dormant) dimeric state of GHR transmembrane domain
5OEK 2018-04-11 Putative active dimeric state of GHR transmembrane domain
1HUW 1994-01-31 2.0 THE CRYSTAL STRUCTURE OF AFFINITY-MATURED HUMAN GROWTH HORMONE AT 2 ANGSTROMS RESOLUTION
1AXI 1998-01-28 2.1 STRUCTURAL PLASTICITY AT THE HGH:HGHBP INTERFACE
1HWG 1997-11-19 2.5 1:2 COMPLEX OF HUMAN GROWTH HORMONE WITH ITS SOLUBLE BINDING PROTEIN
1HGU 1995-12-07 2.5 HUMAN GROWTH HORMONE
1KF9 2002-11-20 2.6 PHAGE DISPLAY DERIVED VARIANT OF HUMAN GROWTH HORMONE COMPLEXED WITH TWO COPIES OF THE EXTRACELLULAR DOMAIN OF ITS RECEPTOR
1A22 1998-04-29 2.6 HUMAN GROWTH HORMONE BOUND TO SINGLE RECEPTOR
2AEW 2005-11-01 2.7 A model for growth hormone receptor activation based on subunit rotation within a receptor dimer
3HHR 1994-04-30 2.8 HUMAN GROWTH HORMONE AND EXTRACELLULAR DOMAIN OF ITS RECEPTOR: CRYSTAL STRUCTURE OF THE COMPLEX
1HWH 1997-11-19 2.9 1:1 COMPLEX OF HUMAN GROWTH HORMONE MUTANT G120R WITH ITS SOLUBLE BINDING PROTEIN
1BP3 1998-08-19 2.9 THE XRAY STRUCTURE OF A GROWTH HORMONE-PROLACTIN RECEPTOR COMPLEX

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
181.4 B,C Somatotropin Q95ML5 GHR_SAIBB
185.4 B,C Somatotropin P79194 GHR_MACMU
190.2 B,C Somatotropin Q9XSZ1 GHR_PAPAN
200.0 B,C Somatotropin P10912 GHR_HUMAN
90.5 A Somatotropin P58343 SOMA_SAIBB
93.2 A Somatotropin P01242 SOM2_HUMAN
90.5 A Somatotropin Q9GMB3 SOMA_CALJA
93.7 A Somatotropin P58757 SOM2_PANTR
96.8 A Somatotropin P33093 SOMA_MACMU
100.0 A Somatotropin P58756 SOMA_PANTR
100.0 A Somatotropin P01241 SOMA_HUMAN