Acid stabilization of human growth hormone equilibrium folding intermediates.


Abstract

Equilibrium denaturation experiments were performed on human growth hormone (hGH) under acidic conditions (pH 1.5-3.0) and different protein concentrations. At 0.1 mg/ml hGH using intrinsic tryptophan fluorescence and far-UV circular dichroism (CD) detection, midpoint values of 4.6 M GdnHCl were observed that are identical to those obtained at neutral pH. However, the delta G values were reduced at pH 2.5 relative to pH 8.0 (10.5 vs. 15 kcal/mol). Increasing the protein concentration to 1 mg/ml resulted in a biphasic denaturation profile by far-UV CD detection at 222 nm, while near-UV CD measurements at 295 nm yielded a cooperative transition with a midpoint value of 3.6 M GdnHCl. These results indicate that equilibrium intermediates having a propensity to aggregate are highly populated under acid conditions. Static light scattering measurements performed under partial unfolding conditions (4.5 M GdnHCl) at pH 2.5 confirmed the existence of a large molecular weight (congruent to 80 kDa) self-associated intermediate. No evidence of aggregation was found for hGH under acid conditions in the absence of denaturant, indicating that self-association results from the formation of an intermediate. Equilibrium GdnHCl concentration-jump experiments confirmed that association only occurs from an intermediate species and not from any other conformational state, and formation of the self-associated intermediate can lead to irreversible loss of protein due to precipitation. These results demonstrate that acid stabilizes equilibrium folding intermediates of hGH. Study holds ProTherm entries: 9361, 9362, 9363, 9364, 9365, 9366, 9367, 9368, 9369 Extra Details: pH; denaturation; intermediates; protein folding;,self-association; growth hormone; (human)

Submission Details

ID: 5ErVsX4B3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:37 p.m.

Version: 1

Publication Details
DeFelippis MR;Kilcomons MA;Lents MP;Youngman KM;Havel HA,Biochim. Biophys. Acta (1995) Acid stabilization of human growth hormone equilibrium folding intermediates. PMID:7873589
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