Kinetic analysis of the folding of human growth hormone. Influence of disulfide bonds.


Abstract

We report the results of a stopped-flow kinetic evaluation of the folding of human growth hormone (hGH). The results are compared with those obtained for a disulfide-modified analog in which the four cysteine residues have been reduced and alkylated to form tetra-S-carbamidomethylated hGH in order to elucidate the role of disulfide bonds in the folding reaction. Multiple detection techniques were applied to monitor both refolding and unfolding processes initiated by guanidine hydrochloride concentration jumps. Using far-UV circular dichroism (CD) detection to monitor folding of hGH, we find that 70% of the secondary structure forms in a burst phase occurring within the stopped-flow dead time. Two slower phases were identified in the observable portion of the CD signal. Multiple kinetic phases were resolved when folding was monitored by intrinsic tryptophan fluorescence or near-UV absorbance as probes of tertiary structure, and the number of time constants required to fit the data depended on the hGH concentration and nature of the denaturant jump. The associated amplitudes also displayed strong dependence on the final denaturant concentration. Results obtained from the tetra-S-carbamidomethylated hGH studies demonstrate that the folding reactions of hGH are remarkably similar in the presence and absence of the disulfide bonds. Disulfide bond reduction in hGH is proposed to affect folding primarily by increasing the population of self-associated intermediate states in the folding pathway. Study holds ProTherm entries: 5200, 5201 Extra Details: cysteine; disulfide bonds; secondary structure;,intermediate states; folding pathway

Submission Details

ID: m8KGbzdt3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:29 p.m.

Version: 1

Publication Details
Youngman KM;Spencer DB;Brems DN;DeFelippis MR,J. Biol. Chem. (1995) Kinetic analysis of the folding of human growth hormone. Influence of disulfide bonds. PMID:7649991
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
5OEK 2018-04-11 Putative active dimeric state of GHR transmembrane domain
5OHD 2018-04-11 Putative inactive (dormant) 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
1HGU 1995-12-07 2.5 HUMAN GROWTH HORMONE
1HWG 1997-11-19 2.5 1:2 COMPLEX OF HUMAN GROWTH HORMONE WITH ITS SOLUBLE BINDING PROTEIN
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